FIELD OF INVENTION The present invention relates to a gene identified in A. actinofnycetemcomitans and the protein encoded by this gene, which induces apoptosis in cells and promotes bacterial adhesion. The invention also relates to the uses of said gene and gene product.

BACKGROUND OF THE INVENTION Human inflammatory and immune responses to specific sub-gingival <BR> <BR> bacterial infections can result in periodontal (gum) disease (i. e. , periodontitis). While heterogeneous in etiology, a common hallmark of periodontitis is alveolar bone destruction, a leading cause of tooth loss in adults (Zambon, 1996). Recently, periodontitis have been shown to be implicated in an increased risk of certain systemic disorders such as congestive heart diseases, stroke, pneumonia and pre-term low birth- weight (Offenbacher, 1996). Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans), a Gram-negative facultative capnophilic rod bacterium, has been identified as the etiological agent of localized juvenile periodontitis (LJP) and some severe forms of adult periodontitis, while being involved in other diseases such as thyroid and brain abscess, urinary tract infections and sub-acute bacterial endocarditis (Zambon et al., 1988). A. actinomycetemcornitans is able to invade the gingival epithelium and releases several virulence factors such as endotoxins and a potent leukotoxin (Fives-Taylor et al., 1999). Periodontal infection by A. actinomycetemcomitans is accompanied by local and systemic antigen-specific immune responses (Ebersole and Taubman, 1994). Studies have shown that A. actihoyzycetemcomitans-associated molecular chaperones such as GroEL-like protein or/and heat shock proteins are cytotoxic and osteolytic to the epithelia and bone, respectively (Kirby et al. 1995; Paju et al., 2000), suggesting that this bacterial species has multiple effects during periodontal infections. Several carbohydrate, fimbria, polysaccharide and protein antigens have been reported (Fives-Taylor et al., 1999; Zambon, 1996). However, less is known about the importance of major antigenic determinants of A. actii2omycetemcomitans that sensitize either T cells or B cells involved in periodontal inflammation and are associated with tissue destruction.

Earlier studies have demonstrated that there were altered CD4/CD8 T cell ratios and autologous mixed lymphocyte reactions in LJP patients (Stoufi et al., 1987; Suzuki et al., 1984) and that T helper cells could home to periodontal tissues in rat and mouse models of periodontitis (Teng et al., 1999; Yamashita et al., 1991). These studies suggest that T cells play a pivotal role in controlling or/and mediating the balance of exacerbation/quiescence of disease progress. Engraftment of immunodeficient NOD/SCID mice with human peripheral blood leukocytes provides an animal model (called HuPBL-NOD/SCID) for studying human immune responses to inoculated antigens/pathogens (Teng et al., 1999). A. actinomycetemcomitans-reactive human CD4+ T-cells were functionally active in HuPBL-NOD/SCID mice (Teng et al., 2000) and their T-cell-receptor repertoire overlapped significantly (>83%) with that of clinical T cell isolates in LJP patients. Further, A. actinomycetemcomitans-reactive human periodontal CD4+ T cells expressed osteoprotegerin-ligand (OPG-L), a key modulator of osteoclastogenesis and osteoclast activation, and in vivo inhibition of OPG-L function by its decoy receptor OPG significantly reduced alveolar bone destruction and the number of osteoclasts at the site of infection (Teng et al., 2000).

Apoptosis is a common mechanism of cell elimination in the multicellular organism. It occurs during embryonic development, and in a variety of normal and malignant adult tissues. Apoptosis can be induced by various signals, such as exposure to corticosteroids, cytotoxic T lymphocytes, tumor necrosis factor alpha (TNFa), some monoclonal antibodies (mAb) and cytotoxic drugs. In addition, apoptosis may be induced by an absence of signaling, such as the deprivation of growth hormone (Arends M. , et al. , 1990; Cotter T. , et al., 1990 ; Gerschenson L. and Rotello R., 1992).

Bacterial cell adhesion is an important component of bacterial. infection.

Molecules which can be used to control cell adhesion, will have many roles, both clinical and non-clinical.

SUMMARY OF THE INVENTION To identify critical microbial antigens of A. actinomycetemcomitans associated with T-cell-mediated alveolar bone destruction in periodontitis, a genomic library of A. actifaomycetemcomitans was screened by expression cloning using disease- associated periodontal CD4+ T-cells from HuPBL-NOD/SCID mice as probes. In accordance with the present invention, one novel gene has been characterized and its. function studied. This gene has been designated as cagE, based on its sequence homology to the cagE gene of Helicobacter pylori (Censini et al., 1996; Tummuru et al., 1995). The cagE gene of A. actinomycetemcomitans encodes a novel protein of 39 kDa, which is manifested as an effector by inducing apoptosis of various cell types.

Further, the protein encoded by the cagE gene is involved in bacterial adherence to the host cells. Thus, CagE is a potent virulence factor, and plays an important role in A. actinomycetemcomitans-associated diseases.

In accordance with one embodiment of the invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding a CagE ; protein from A. actinomycetemcomitans. In accordance with another embodiment of the invention, there is provided an isolated CagE protein fromA. actinoiycetemcomitans.

This invention is directed to a method of using the cagE gene or a fragment thereof as a diagnostic to screen for the presence of A. actinomycetemcomitans infection. This invention is also directed to a method of using the CagE protein or a fragment thereof as a diagnostic to screen for the presence of A. actinomycetemcomitans infection.

This invention is also directed to a method of using the CagE protein or a fragment thereof for the preparation of a vaccine to either enhance, amplify or desensitize the immune/inflammatory responses in order to modulate and/or shut down CagE-induced apoptosis from A. actinomycetemcomitans infection. The invention also relates to the vaccines so produced.

This invention is directed to a method of using the CagE protein or a fragment thereof for the preparation of antibodies, including polyclonal antibodies, monoclonal antibodies or functional fragment thereof. This invention also relates to the antibodies so produced.

This invention is also directed to vaccines prepared from said antibodies or CagE protein or a fragment thereof, for the treatment or prevention of A. actinomycetemcomitans mediated diseases.

This invention is directed to a method of using said antibodies or a fragment thereof as a diagnostic to screen for the presence of A. actirzomycetemcomitans infection.

This invention is directed to a method of using the CagE protein or a fragment thereof as a drug development tool.

The invention is further directed to a method of modulating apoptosis comprising administering to a cell or animal in need thereof, an effective amount of agent that modulates CagE expression and/or activity.

The invention is also directed to a method of modulating cell adhesion comprising administering to a cell or animal in need thereof, an effective amount of agent that modulates CagE expression and/or activity.

The invention is yet further directed to a method of modulating a condition associated with A. actinomycetemcomitans comprising of administering to a

cell or animal in need thereof, an effective amount of agent that modulates CagE expression and/or activity.

A method of identifying microbial antigens of A. actinomycetemcomitans associated with T-cell-mediated alveolar bone destruction in periodontitis is also part of the present invention.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein: Figures 1A-B are photographs showing the screening of a screening of A. actifaomycetemcofnitans genomic library. Figure 1A photograph shows the identification of the positive clones by screening a genomic library of A. actinofnycetemcornitans through expression cloning using periodontal CD4+ T cells (obtained from Aa-inoculated HuPBL-NOD/SCID mice ; Teng et al., 2000) transfected with cxl2 vector containing hIL-2 promoter and lacZ sequences in vitro. This photograph shows the activated T cells with blue-coloured staining after overnight culture and X-gal treatment in vitro (shown in the drawing as dark colonies). Figure 1B represents the results of a control experiment of Figure 1A using cxl2 vector only without hIL-2 promoter and lacZ sequences.

Figures 2A-D show the cloning of the cagE gene encoding a putative type-IV secretion protein in A. actinomycetemcomita7ls. Figure 2A shows recombinant CagE protein expressed in E. coli as assessed by Western blot using an anti-6His tag antibody (1: 1000 dilution). Induced or un-induced E. coli cell lysate and purified CagE protein were analyzed. E. coli lysate was run at 5 ug/lane and purified protein at 0.5 Mg/lane. Figure 2B shows the sequence alignment of CagE protein (Aa-CagE) (SEQ ID NO: 2) and its homologues, H. pylori CagE (Hp-CagE) (SEQ ID NO: 3) and A. tumefacie71s VirB4 (A-VirB4) (SEQ ID NO: 4). Identical amino acids are shown in shading. Figure 2C shows the nucleic acid sequence (SEQ ID NO: 1) and the amino acid sequence (SEQ ID NO: 2) of the CagE gene of A. actinofnycetemcomitans. Figure

2D shows the full-length PCR transcripts, 1 kb in size, of the cagE gene were amplified from various A. actinontycetenicomitans strains using the same pair of primers.

Figures 3A-B show an immunoblot analysis of the CagE protein expression in various A. actinomycetemcomitans strains. Figure 3A shows that a polyclonal mouse serum raised against recombinant CagE reacted specifically with recombinant CagE protein. The mouse serum was diluted 1: 500 for use. Figure 3B shows that native CagE proteins produced from various A. actinomycetemcomitans (Aa) strains were detected by Western blotting using anti-CagE Ab (1: 500 dilution). 5 Mg of protein/lane were loaded and 0. 5 pg of purified CagE protein was used as a control.

Figures 4A-B show the clinical significance of the CagE protein in A. actinomycetemcomitans-infected patients. Figure 4A shows by ELISA, that all 5 patients' (JP1-JP4 and AP) sera, but not serum from the healthy control (HC) subject, showed high titers of IgG responses to recombinant CagE protein. CagE protein was coated at 101lg/rnl for ELISA. Individual serum samples were diluted 1: 100 and beef insulin (BI) protein was used as the control for specificity in ELISA. Figure 4B shows by Western blot, that all 5 patients'sera, but not serum from healthy control (HC) subject, readily recognized the CagE protein by immunoblot analysis. Individual serum samples were diluted 1: 50 before use.

Figures 5A-C show recombinant CagE protein induces apoptosis of various human cells. Figure 5A shows that typical changes in cellular morphology for apoptosis were observed in CagE-treated (0. 5pm) human primary cells within 6-12 h (right panel), but not in untreated control cells (left panels). From top to bottom in Figure 5A includes: human primary epithelial cells prepared from gingival discards during periodontal surgeries, human epithelial KB cell line, human primary T cells prepared from peripheral blood, human primary osteoblasts and human primary endothelial cells prepared from lung micro-vasculature. Figure 5B shows that DNA fragmentation was detected in CagE-treated (0. 5pm) human primary T cells, primary osteoblasts and KB epithelial cells at 6-12 hrs as indicated, but not in un-treated, or OVA-treated controls. The results obtained from CagE-treated primary lung endothelial cells in Figure 5A were the same thus not shown here. Figure 5C shows cellular cytotoxicity of CagE protein at indicated concentrations on human KB epithelial cells.

Cytotoxicity was assayed in vitro (24h) as described in Example 3. The OVA was used as a control antigen.

Figures 6A-D show that the cagE mutant fails to induce apoptosis of human epithelial cells. Figure 6A shows immunoblot analysis showing the absence of CagE protein in both culture supernatant (SN) and cell lysate of the cagE mutant, compared to the wild type (WT) JP2 strain. Figure 6B shows analysis of the DNA

fragmentation on KB cells treated with WT JP2 strain and cagE-mutant (Bacteria-to- KB ratio: 50,000 to 1). Note that DNA ladder was only detected in WT JP2-treated KB cells, but not in cagE-mutant-treated KB cells. Figure 6C shows TUNEL analysis of KB cells treated with WT JP2 strain and cagE-mutant. Note that significantly more TUNEL-positive green-fluorescent cells were detected in WT JP2-treated KB cells at 4 hrs, than in cagE mutant-treated KB cells. Figure 6D shows that cytochalasin D treatment did not change the presence and absence of apoptotic KB cells induced by either WT JP2 or cagE mutant strains in vitro by 4 hrs, respectively. The results shown here used cytochalasin D of 1 ug/ml. Varying the concentrations of cytochalasin D from 0.5 to 2 gg/n-d in vitro did not alter the results obtained.

Figures 7A-B show that CagE is involved in bacterial adherence to human epithelial cells. Figure 7A shows B/W photographs (40x magnifications) showing abundant contacts of WT JP2 strain on the KB cell surfaces (opaque areas representing bacteria adhering to KB cells), with significantly less contacts made by cagE-mutant. Figure 7B shows photographs (100x magnifications) showing the phase- contrast images of (A) with black dots representing bacterial contacts on the cell surfaces by WT JP2 and cagE-mutant, respectively.

Figure 8 shows that the N-terminal region (1-136) of the CagE is critically involved in inducing apoptosis. Top panels: Typical changes in cellular morphology for apoptosis were observed in Cl-CagE (SEQ ID NO: 5) (CagE with amino acids 310-340 removed) treated (0. 5) im) KB cells in 4hrs, but not in N-CagE (SEQ ID NO: 6) (CagE with amino acids 1-136 removed) treated KB cells. Middle panels: There were significantly more TUNEL-positive signals detected in Cl-CagE treated than those in N-CagE treated KB cells at 4hrs in vitro. Lower panels: A significantly higher amount of TUNEL-positive apoptotic KB cells was detected in 4hrs co-culture with Cl-CagE proteins than that with N-CagE. UV-treated KB culture was used as positive control.

Proteins concentrations used were 0. 5, um. The results of using whole CagE vs. C2- CagE (SEQ ID NO: 7) truncated proteins (CagE with amino acids 271-340 removed) were similar to those of using Cl-CagE proteins shown here (data not shown).

Figure 9A-C show truncated sequences of Aa-cagE. Figure 9A shows the sequence representing an N-terminal truncated construct of the Aa-cagE gene where N-terminal nucleotides 1-408 of the gene have been removed (SEQ ID NO: 8). Figure 9B shows the sequence representing the Cl-terminal truncated construct of the Aa-cagE gene where C-terminal nucleotides 930-1020 of the gene have been removed (SEQ ID NO: 9). Figure 9C shows the sequence representing the C2-terminal truncated construct of the Aa-cagE gene where C-terminal nucleotides 813-1020 of the gene have been removed (SEQ ID NO: 10).

DESCRIPTION OF PREFERRED EMBODIMENT I. Nucleic Acid Molecules of the Invention The present invention relates to a gene identified in A. actinomycetemcomitans and the protein encoded by this gene, which induces apoptosis in cells and promotes bacterial adhesion.

Periodontal CD4+ T cells in A. actinomycetemcomitans-inoculated HuPBL-NOD/SCID mice have been shown to be associated with tissue inflammation and alveolar bone destruction in vivo (Teng et al., 1999; Teng et al., 2000). By using the periodontal CD4+ T cells, after expanding in vitro, as probes to screen the genomic DNA library of A. actinomycetemcomitans, bacterial antigens of A. actinomycetemcomitans involved in the above process were identified. Thus, according to one aspect of the present invention there is provided a method of of identifying microbial antigens of A. actinomyceterncomitans associated with T-cell-mediated alveolar bone destruction in periodontitis comprising the steps of : growing individual E. coli clones in a suitable media; exposing the clones to disease-associated periodontitis CD4+cells transfected with a suitable reporter gene; identifying clones that induced T cell activation by use of said reporter gene; and identifying the microbial antigen from said clones.

More than 6 different genomic clones of A. actinomycetemcomtans were identified (data not shown). After comparing the GenBank sequences available, one of the positive clones identified (F023) was shown to contain a partial DNA sequence with significant homology to the cagE gene of H. pylori, originally described as part of the bacterial type-IV secretion system (Censini et al., 1996; Tummuru et al., 1995).

Through a search of the sequencing data of A. actinomycetemcomitans genome project at the Oklahoma University (www. genome. ou. edu/act. html), a putative open reading frame (ORF) encoding a protein of 340 residues, with a predicted MW of 38.6 kDa, hereafter designated CagE was identified. The cagE gene ORF was cloned into pQE30 expression vector and the recombinant CagE protein was then expressed in E. coli.

Western blot analysis showed that the recombinant CagE protein had a molecular weight of 39 kDa, which is consistent with the predicted molecular weight.

The deduced amino acid (a. a. ) sequence of A. actinomycetemcomitans CagE protein showed significant homology to those of H. pylori CagE and Agrobacterium tumefaciens (A. tumefaciens) VirB4, both of which are encoded by a type-IV secretion system associated with virulence or pathogenesis in their respective hosts (Censini et al., 1996; Tummuru et al., 1995; Covacci et al., 1999; Chrisitie and Vogel, 2000; Covacci and Rappuoli, 2000). The CagE protein of A. actinomycetemcomitans is shorter (only 340 a. a), than its counterparts H. pylori CagE

(981 a. a) and A. tumefaciens VirB4 (789 a. a). Sequence alignment showed that CagE protein in A. actinomycetemcomitans shared high homology to the C-termini (after residue 300) of its counterparts. Specifically, a. a. residues of A. actinomycetemcomitans CagE were 29% identical to those of H. pylori CagE and 21% to A. tumefaciens VirB4. In addition, A. actinomycetemcomitans CagE also showed significant homology (about 20% identical) to VirB4 proteins of Bordetella pertussis, Bartonella henselae and Brucella aborts, an invasion-associated protein (Opc) from Neisseria meningitidis, and TraB protein of E. coli (review: Chrisitie and Vogel, 2000; data not shown).

To further investigate whether there are any putative cag or cag-like type- IV secretion gene family (i. e., cag Pathogenecity Island-PAI) in A. actinomycetemcomitans, genomic DNA fragment (of HK 1651 strain) flanking the cagE gene was cloned and analyzed by genomic DNA walking and sequencing. The results showed that, within ~6 kb flanking region around the cagE gene, there are two more putative ORFs showing homology to the cagD and cagH genes of H. pylori (Censini et al., 1996; data not shown). More specifically, the cagD (or cagD-like) homologue (25% identity) is located (-3kb) at the 5'-end of the cagE gene while the cagH (or cagH-like) homologue (19% identity) is located (-lkb) at the 3'-end of the cagE gene. In addition, a further search of the A. actinomycetemcomitans genomic sequence database (www. genome. ou. edu/act. html) revealed a few more putative ORFs carrying homology to the bacterial type-IV secretion proteins, including CagC and CagM of H. pylori ; VirB 1 and VirB 11 of A. tumefaciens (sequence alignment not shown).

Therefore the present invention provides an isolated nucleic acid molecule from A. actinomycetemcomitans comprising a sequence encoding a CagE protein having a molecular weight of approximately 39 kDa.

The term"isolated"refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. The term "nucleic acid"is intended to include DNA and RNA and can be either double stranded or single stranded.

In an embodiment of the invention, an isolated nucleic acid molecule is provided having a sequence which encodes a CagE protein having the amino acid sequence as shown in Figure 2C (SEQ ID NO: 2).

In a preferred embodiment, the invention provides an isolated nucleic acid sequence comprising: (a) a nucleic acid sequence as shown in Figure 2C (SEQ ID NO: 1);

(b) a nucleic acid sequence that is complimentary to a nucleic acid sequence of (a); (c) a nucleic acid sequence that has substantial sequence homology to a nucleic acid sequence of (a) or (b); (d) a nucleic acid sequence that is an analog of a nucleic acid sequence of (a), (b) or (c); or (e) a nucleic acid sequence that hybridizes to a nucleic acid sequence of (a), (b), (c) or (d) under stringent hybridization conditions.

The term"sequence that has substantial sequence homology"means those nucleic acid sequences which have slight or inconsequential sequence variations <BR> <BR> from the sequences in (a) or (b), i. e. , the sequences function in substantially the same manner and can be used for example to modulate apoptosis or cell adhesion. The variations may be attributable to local mutations or structural modifications. Nucleic acid sequences having substantial homology include nucleic acid sequences having at least 65%, preferably at least 75%, more preferably at least 85%, and most preferably 90-95% identity with the nucleic acid sequences as shown in Figure 2C (SEQ ID NO: 1). DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions are within the skill of one in the art (see for example Maniatis et al., 1982 and Sambrook et al. 1989).

The term"sequence that hybridizes"means a nucleic acid sequence that can hybridize to a sequence of (a), (b), (c) or (d) under stringent hybridization conditions. Appropriate"stringent hybridization conditions"which promote DNA hybridization are known to those skilled in the art, or may be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3. 1-6.3. 6. For example, the following may be employed: 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C ; 0.2 x SSC at 50°C to 65°C ; or 2.0 x SSC at 44°C to 50°C. The stringency may be selected based on the conditions used in the wash step. For example, the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature in the wash step can be at high stringency conditions, at about 65°C.

The term"a nucleic acid sequence which is an analog"means a nucleic acid sequence which has been modified as compared to the sequence of (a), (b) or (c) wherein the modification does not alter the utility of the sequence as described herein.

The modified sequence or analog may have improved properties over the sequence shown in (a), (b) or (c). One example of a modification to prepare an analog is to replace one of the naturally occurring bases (i. e. adenine, guanine, cytosine or

thymidine) of the sequence shown in Figure 2C (SEQ ID NO: 1), with a modified base such as such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8- thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8 amino guanine, 8-thiol guanine,, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.

Another example of a modification is to include modified phosphorous or oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages in the nucleic acid molecule shown in Figure 2C (SEQ ID NO: 1). For example, the nucleic acid sequences may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates.

A further example of an analog of a nucleic acid molecule of the invention is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P. E. Nielsen, et al Science 1991,254, 1497). PNA analogs have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. PNAs also bind more strongly to a complimentary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand. Other nucleic acid analogs may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones. For example, the nucleotides may have morpholino backbone structures (U. S. Pat. No. 5, 034,506). The analogs may also contain groups such as reporter groups, a group for improving the pharmacokinetic or pharmacodynamic properties of nucleic acid sequence.

It will be appreciated that the invention includes nucleic acid molecules encoding truncations of proteins of the invention, and analogs and homologs of proteins of the invention and truncations thereof, as described below. It will further be appreciated that variant forms of nucleic acid molecules of the invention which arise by alternative splicing of an mRNA corresponding to a cDNA of the invention are encompassed by the invention.

Isolated and purified nucleic acid molecules having sequences which differ from the nucleic acid sequence of the invention due to degeneracy in the genetic code are also within the scope of the invention. Such nucleic acids encode functionally equivalent proteins but differ in sequence from the above mentioned sequences due to degeneracy in the genetic code.

An isolated nucleic acid molecule of the invention which comprises DNA can be isolated by preparing a labelled nucleic acid probe based on all or part of the nucleic acid sequences of the invention and using this labelled nucleic acid probe to screen an appropriate DNA library (e. g. a cDNA or genomic DNA library). For example, a genomic library isolated can be used to isolate a DNA encoding a novel protein of the invention by screening the library with the labelled probe using standard techniques. Nucleic acids isolated by screening of a cDNA or genomic DNA library can be sequenced by standard techniques.

An isolated nucleic acid molecule of the invention which is DNA can also be isolated by selectively amplifying a nucleic acid encoding a novel protein of the invention using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic oligonucleotide primers from the nucleic acid sequence of the invention for use in PCR. A nucleic acid can be amplified from cDNA or genomic DNA using these oligonucleotide primers and standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. It will be appreciated that cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al., Biochemistry, 18,5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc. , St. Petersburg, FL).

An isolated nucleic acid molecule of the invention which is RNA can be isolated by cloning a cDNA encoding a novel protein of the invention into an appropriate vector which allows for transcription of the cDNA to produce an RNA molecule which encodes a protein of the invention. For example, a cDNA can be cloned <BR> <BR> downstream of a bacteriophage promoter, (e. g. , a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by standard techniques.

A nucleic acid molecule of the invention may also be chemically synthesized using standard techniques. Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See <BR> <BR> e. g. , Itakura et al. U. S. Patent No. 4,598, 049; Caruthers et al. U. S. Patent No. 4,458, 066; and Itakura U. S. Patent Nos. 4,401, 796 and 4,373, 071).

Determination of whether a particular nucleic acid molecule encodes a novel protein of the invention may be accomplished by expressing the cDNA in an

appropriate host cell by standard techniques, and testing the activity of the protein using the methods as described herein. A cDNA having the activity of a novel protein of the invention so isolated can be sequenced by standard techniques, such as dideoxynucleotide chain termination or Maxam-Gilbert chemical sequencing, to determine the nucleic acid sequence and the predicted amino acid sequence of the encoded protein.

The initiation codon and untranslated sequences of nucleic acid molecules of the invention may be determined using currently available computer <BR> <BR> software designed for the purpose, such as PC/Gene (IntelliGenetics Inc. , Calif.).

Regulatory elements can be identified using conventional techniques. The function of the elements can be confirmed by using these elements to express a reporter gene which is operatively linked to the elements. These constructs may be introduced into cultured cells using standard procedures. In addition to identifying regulatory elements in DNA, such constructs may also be used to identify proteins interacting with the elements, using techniques known in the art.

The sequence of a nucleic acid molecule of the invention may be inverted relative to its normal presentation for transcription to produce an antisense nucleic acid molecule which are more fully described herein. Preferably, an antisense sequence is constructed by inverting a region preceding the initiation codon or an unconserved region. In particular, the nucleic acid sequences contained in the nucleic acid molecules of the invention or a fragment thereof, may be inverted relative to its normal presentation for transcription to produce antisense nucleic acid molecules. Antisense molecules are further described below.

The invention also provides nucleic acids encoding fusion proteins comprising a novel protein of the invention and a selected protein, or a selectable marker protein.

Also provided are portions of the nucleic acid sequence encoding fragments, functional domains or antigenic determinants of the CagE protein. The present invention also provides for the use of portions of the sequence as probes and PCR primers for CagE and related proteins as well as for determining functional aspects of the sequence.

One of ordinary skill in the art is now enabled to identify and isolate CagE genes or cDNAs which are allelic variants of the disclosed CagE sequence, using standard hybridization screening or PCR techniques.

II. Novel Proteins of the Invention The invention further broadly contemplates an isolated CagE protein having a molecular weight of approximately 40 KDa. The term"CagE protein"as

used herein includes all homologs, analogs, fragments or derivatives of the CagE protein from A. actinomycetemcomitans.

In one embodiment, the isolated CagE has an amino acid sequence as shown in Figure 2C (SEQ ID NO: 2).

Within the context of the present invention, a protein of the invention may include various structural forms of the primary protein which retain biological activity. For example, a protein of the invention may be in the form of acidic or basic salts or in neutral form. In addition, individual amino acid residues may be modified by oxidation or reduction.

In addition to the full length amino acid sequence, the protein of the present invention may also include truncations of the protein, and analogs, and homologs of the protein and truncations thereof as described herein. Truncated proteins or fragments may comprise peptides of at least 5, preferably 10 and more preferably 15 amino acid residues of the sequence shown in Figure 2C (SEQ ID NO: 2). Examples of truncated CagE protein include Cl-CagE (SEQ ID NO: 5), N-CagE (SEQ ID NO: 6) and C2-CagE (SEQ ID NO : 7) which are further described in Example 6.

The invention further provides polypeptides comprising at least one functional domain or at least one antigenic determinant of a CagE protein.

Analogs of the protein of the invention and/or truncations thereof as described herein, may include, but are not limited to an amino acid sequence containing one or more amino acid substitutions, insertions, and/or deletions. Amino acid substitutions may be of a conserved or non-conserved nature. Conserved amino acid substitutions involve replacing one or more amino acids of the proteins of the invention with amino acids of similar charge, size, and/or hydrophobicity characteristics. When only conserved substitutions are made the resulting analog should be functionally equivalent. Non-conserved substitutions involve replacing one or more amino acids of the amino acid sequence with one or more amino acids which possess dissimilar charge, size, and/or hydrophobicity characteristics.

One or more amino acid insertions may be introduced into the amino acid sequences of the invention. Amino acid insertions may consist of single amino acid residues or sequential amino acids ranging from 2 to 15 amino acids in length. For example, amino acid insertions may be used to destroy target sequences so that the protein is no longer active. This procedure may be used in vivo to inhibit the activity of a protein of the invention.

Deletions may consist of the removal of one or more amino acids, or discrete portions from the amino acid sequence of the CagE. The deleted amino acids

may or may not be contiguous. The lower limit length of the resulting analog with a deletion mutation is about 10 amino acids, preferably 100 amino acids.

Analogs of a protein of the invention may be prepared by introducing mutations in the nucleotide sequence encoding the protein. Mutations in nucleotide sequences constructed for expression of analogs of a protein of the invention must preserve the reading frame of the coding sequences. Furthermore, the mutations will preferably not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, which could adversely affect translation of the receptor mRNA.

Mutations may be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site specific mutagenesis procedures may be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Deletion or truncation of a protein of the invention may also be constructed by utilizing convenient restriction endonuclease sites adjacent to the desired deletion. Subsequent to restriction, overhangs may be filled in, and the DNA related. Exemplary methods of making the alterations set forth above are disclosed by Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory Press, 1989).

The proteins of the invention also include homologs of the amino acid sequence of the CagE protein and/or truncations thereof as described herein. Such homologs are proteins whose amino acid sequences are comprised of amino acid sequences that hybridize under stringent hybridization conditions (see discussion of stringent hybridization conditions herein) with a probe used to obtain a protein of the invention. Homologs of a protein of the invention will have the same regions which are characteristic of the protein.

A homologous protein includes a protein with an amino acid sequence having at least 60%, preferably at least 70%, more preferably 80-95% identity with the amino acid sequence of the CagE protein.

The invention also contemplates isoforms of the proteins of the invention. An isoform contains the same number and kinds of amino acids as a protein of the invention, but the isoform has a different molecular structure. The isoforms contemplated by the present invention are those having the same properties as a protein of the invention as described herein.

The present invention also includes a protein of the invention conjugated with a selected protein, or a selectable marker protein to produce fusion proteins. For example, the CagE cDNA sequence is inserted into a vector that contains a nucleotide sequence encoding another peptide (e. g. GST-glutathione succinyl transferase). The fusion protein is expressed and recovered from prokaryotic (e. g. bacterial or baculovirus) or eukaryotic cells. The fusion protein can then be purified by affinity chromatography based upon the fusion vector sequence and the CagE protein obtained by enzymatic cleavage of the fusion protein. <BR> <BR> <P>The proteins of the invention (including truncations, analogs, etc. )<BR> be prepared using recombinant DNA methods. Accordingly, nucleic acid molecules of the present invention having a sequence which encodes a protein of the invention may be incorporated according to procedures known in the art into an appropriate expression vector which ensures good expression of the protein. Possible expression vectors <BR> <BR> include but are not limited to cosmids, plasmids, or modified viruses (e. g. ,<BR> defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used. The expression"vectors suitable for transformation of a host cell", means that the expression vectors contain a nucleic acid molecule of the invention and regulatory sequences, selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule.

"Operatively linked"is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid.

The invention therefore contemplates a recombinant expression vector of the invention containing a nucleic acid molecule of the invention, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, or viral genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Selection of appropriate regulatory sequences is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include : a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal.

Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. It will also be appreciated that the necessary regulatory sequences may be supplied by the native protein and/or its flanking regions.

The invention further provides a recombinant expression vector comprising a DNA nucleic acid molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression, by transcription of the DNA molecule, of an RNA molecule which is antisense to a nucleotide sequence of the invention. Regulatory sequences operatively linked to the antisense nucleic acid can be chosen which direct the continuous expression of the antisense RNA molecule.

The recombinant expression vectors of the invention may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention. Examples of selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, p-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as P-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of recombinant expression vectors of the invention and in particular to determine the effect of a mutation on expression and phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest.

The recombinant expression vectors may also contain genes which encode a fusion moiety which provides increased expression of the recombinant protein; increased solubility of the recombinant protein; and aid in the purification of a target recombinant protein by acting as a ligand in affinity purification. For example, a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.

Recombinant expression vectors can be introduced into host cells to produce a transformed host cell. Accordingly, the invention includes a host cell comprising a recombinant expression vector of the invention. The term"transformed host cell"is intended to include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention. The <BR> <BR> terms"transformed with", "transfected<BR> intended to encompass introduction of nucleic acid (e. g. a vector) into a cell by one of many possible techniques known in the art. Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium-chloride mediated

transformation. Nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE- dextran-mediated transfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al.

(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989) ), and other such laboratory textbooks.

Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins of the invention may be expressed in bacterial cells such as E. coli, Pseudomonas, Bacillus subtillus, insect cells (using baculovirus), yeast cells or mammalian cells. Other suitable host cells can be found in Goeddel, Gene Expression Technology : Methods in Enzymology 185, Academic Press, San Diego, CA (1991).

As an example, to produce CagE proteins recombinantly, for example, E. coli can be used using the T7 RNA polymerase/promoter system using two plasmids or by labeling of plasmid-encoded proteins, or by expression by infection with M13 Phage mGPI-2. E. coli vectors can also be used with Phage lamba regulatory sequences, by fusion protein vectors (e. g. lacZ and trpE), by maltose-binding protein fusions, and by glutathione-S-transferase fusion proteins.

Alternatively, the CagE protein can be expressed in insect cells using baculoviral vectors, or in mammalian cells using vaccinia virus. For expression in mammalian cells, the cDNA sequence may be ligated to heterologous promoters, such as the simian virus (SV40) promoter in the pSV2 vector and introduced into cells, such as COS cells to achieve transient or long-term expression. The stable integration of the chimeric gene construct may be maintained in mammalian cells by biochemical selection, such as neomycin and mycophoenolic acid.

The CagE DNA sequence can be altered using procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, site-directed sequence alteration with the use of specific oligonucleotides together with PCR.

The cDNA sequence or portions thereof, or a mini gene consisting of a cDNA with an intron and its own promoter, is introduced into eukaryotic expression vectors by conventional techniques. These vectors permit the transcription of the cDNA in eukaryotic cells by providing regulatory sequences that initiate and enhance the transcription of the cDNA and ensure its proper splicing and polyadenylation. The endogenous CagE gene promoter can also be used. Different promoters within vectors have different activities which alters the level of expression of the cDNA. In addition,

certain promoters can also modulate function such as the glucocorticoid-responsive promoter from the mouse mammary tumor virus.

Some of the vectors listed contain selectable markers or neo bacterial genes that permit isolation of cells by chemical selection. Stable long-term vectors can be maintained in cells as episomal, freely replicating entities by using regulatory elements of viruses. Cell lines can also be produced which have integrated the vector into the genomic DNA. In this manner, the gene product is produced on a continuous basis.

Vectors are introduced into recipient cells by various methods including calcium phosphate, strontium phosphate, electroporation, lipofection, DEAE dextran, microinjection, or by protoplast fusion. Alternatively, the cDNA can be introduced by infection using viral vectors.

CagE proteins may also be isolated from A. actinomycetemcomitans, in which the protein is normally expressed.

The protein may be purified by conventional purification methods known to those in the art, such as chromatography methods, high performance liquid chromatography methods or precipitation.

For example, an anti-CagE antibody (as described below) may be used to isolate a CagE protein, which is then purified by standard methods.

The proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85: 2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E.

Wansch, Vol. 15 I and IT, Thieme, Stuttgart).

III. Uses The present invention includes all uses of the nucleic acid molecules and CagE proteins of the invention including, but not limited to, the preparation of all forms of antibodies and antisense oligonucleotides, the isolation of substances that modulate CagE expression and/or activity as well as the use of the CagE nucleic acid sequences and proteins and modulators thereof in diagnostic, biomedical, therapeutic and vaccine applications. Some of the uses are further described below.

(a) Antibodies The isolation of the CagE protein enables the preparation of antibodies specific for CagE from A. actiizomycetey7zcomitans. Accordingly, the present invention provides an antibody that binds to a CagE protein from A. actiyaomycetemcomitans.

Antibodies can be prepared which bind a distinct epitope in an unconserved region of

the protein. An unconserved region of the protein is one that does not have substantial sequence homology to other proteins.

Antibodies to a CagE protein may be prepared as described in Examples 2 and 7. Antibodies to CagE may also be prepared using techniques known in the art.

For example, by using a peptide of CagE, polyclonal antisera or monoclonal antibodies <BR> <BR> can be made using standard methods. A mammal, (e. g. , a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the peptide which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art. For example, the protein or peptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum.

Standard ELISA or other immunoassay procedures can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.

To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art, (e. g., the hybridoma technique originally developed by Kohler and Milstein (Nature 256,495-497 <BR> <BR> (1975) ) as well as other techniques such as the human B-cell hybridoma technique<BR> (Kozbor et al., Immunol. Today 4,72 (1983) ), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al. Monoclonal Antibodies in Cancer <BR> <BR> Therapy (1985) Allen R. Bliss, Inc. , pages 77-96), and screening of combinatorial<BR> antibody libraries (Huse et al., Science 246,1275 (1989) ). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the peptide and the monoclonal antibodies can be isolated. Therefore, the invention also contemplates hybridoma cells secreting monoclonal antibodies with specificity for CagE as described herein.

The term"antibody"as used herein is intended to include fragments thereof which also specifically react with CagE from A. actinomycetemcomitans, or fragments thereof. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above. For example, F (ab') 2 fragments can be generated by treating antibody with pepsin. The resulting F (ab') 2 fragment can be treated to reduce disulfide bridges to produce Fab'fragments.

Chimeric antibody derivatives, i. e. , antibody molecules that combine a non-human animal variable region and a human constant region are also contemplated within the scope of the invention. Chimeric antibody molecules can include, for

example, the antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions. Conventional methods may be used to make chimeric antibodies containing the immunoglobulin variable region which recognizes the gene product of CagE antigens of the invention (See, for example, Morrison et al. , Proc. Natl<BR> Acad. Sci. U. S. A. 81,6851 (1985); Takeda et al. , Nature 314,452 (1985), Cabilly et<BR> al. , U. S. Patent No. 4,816, 567; Boss et al., U. S. Patent No. 4,816, 397; Tanaguchi et<BR> al. , European Patent Publication EP171496 ; European Patent Publication 0173494, United Kingdom patent GB 2177096B). It is expected that chimeric antibodies would be less immunogenic in a human subject than the corresponding non-chimeric antibody.

Monoclonal or chimeric antibodies specifically reactive with a protein of the invention as described herein can be further humanized by producing human constant region chimeras, in which parts of the variable regions, particularly the conserved framework regions of the antigen-binding domain, are of human origin and only the hypervariable regions are of non-human origin. Such immunoglobulin molecules may be made by techniques known in the art, (e. g. , Teng et al., Proc. Natl.<BR> <P>Acad. Sci. U. S. A. , 80,7308-7312 (1983); Kozbor et al., Immunology Today, 4,7279<BR> (1983); Olsson et al. , Meth. Enzymol. , 92,3-16 (1982) ), and PCT Publication W092/06193 or EP 0239400). Humanized antibodies can also be commercially produced (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.) Specific antibodies, or antibody fragments, such as, but not limited to, single-chain Fv monoclonal antibodies reactive against CagE proteins may also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with peptides produced from the nucleic acid molecules of CagE. For example, complete Fab fragments, VH regions and FV regions can be expressed in bacteria using phage expression libraries (See for example Ward et al., Nature 341,544-546 : (1989); Huse et al., Science 246,1275-1281 (1989); and McCafferty et al. Nature 348,552-554 (1990) ). Alternatively, a SCID-hu mouse, for example the model developed by Genpharm, can be used to produce antibodies or fragments thereof.

(b) Antisense Oligonucleotides Isolation of a nucleic acid molecule encoding CagE enables the production of antisense oligonucleotides that can modulate the expression and/or activity of CagE.

Accordingly, the present invention provides an antisense oligonucleotide that is complimentary to a nucleic acid sequence encoding CagE.

The term"antisense oligonucleotide"as used herein means a nucleotide sequence that is complimentary to its target.

The term"oligonucleotide"refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted oligonucleotides may be preferred over naturally occurring forms because of properties such as enhanced cellular uptake, or increased stability in the presence of nucleases. The term also includes chimeric oligonucleotides which contain two or more chemically distinct regions. For example, chimeric oligonucleotides may contain at least one region of modified nucleotides that confer beneficial properties (e. g. increased nuclease resistance, increased uptake into cells), or two or more oligonucleotides of the invention may be joined to form a chimeric oligonucleotide.

The antisense oligonucleotides of the present invention may be ribonucleic or deoxyribonucleic acids and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The oligonucleotides may also contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8- hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosine.

Other antisense oligonucleotides of the invention may contain modified phosphorous, oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. For example, the antisense oligonucleotides may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates. In an embodiment of the invention there are phosphorothioate bonds links between the four to six 3'-terminus bases. In another embodiment phosphorothioate bonds link all the nucleotides.

The antisense oligonucleotides of the invention may also comprise nucleotide analogs that may be better suited as therapeutic or experimental reagents. An example of an oligonucleotide analogue is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P. E. Nielsen, et al Science 1991,254, 1497). PNA analogues have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. PNAs also bind

more strongly to a complimentary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand. Other oligonucleotides may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones. For example, the nucleotides may have morpholino backbone structures (U. S. Pat. Nol 5,034, 506). Oligonucleotides may also contain groups such as reporter groups, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an antisense oligonucleotide. Antisense oligonucleotides may also have sugar mimetics.

The antisense nucleic acid molecules may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. The antisense nucleic acid molecules of the invention or a fragment thereof, may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene e. g. phosphorothioate derivatives and acridine substituted nucleotides. The antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.

The antisense oligonucleotides may be introduced into tissues or cells using techniques in the art including vectors (retroviral vectors, adenoviral vectors and DNA virus vectors) or physical techniques such as microinjection. The antisense oligonucleotides may be directly administered in vivo or may be used to transfect cells in vitro which are then administered in vivo. In one embodiment, the antisense oligonucleotide may be delivered to macrophages and/or endothelial cells in a liposome formulation.

(c) Diagnostic Assay The finding by the present inventors that CagE is involved in the virulence of A. actinomycetemcomitans allows the development of diagnostic assays for A. actinomycetenicomitans.

Four lines of evidence suggests that CagE may be a very important virulence factor in A. actinomycetemco2itans : i) this gene was repeatedly identified (in 75% of the patients tested) from screening by expression cloning using disease- associated periodontal CD4+ T cells derived from HuPBL-NOD/SCID mice as probes; ii) all serum samples from five A. actiyzomycetemcomitans-infected periodontitis patients readily recognized CagE protein by ELISA and Western blot; iii) the recombinant CagE

protein at pM concentrations induced apoptosis of various human cells; and iv) cagE mutant failed to induce apoptosis of human epithelial cells and showed significant reduction of its adherence to the host cells.

The function of bacterial type-IV secretion system has been shown primarily to mobilize DNA from bacteria to bacteria or toxic protein (s) from bacteria to eukaryotic cells (Covacci et al., 1999; Chrisitie and Vogel, 2000; Covacci and Rappuoli, 2000). The cag gene family was first described in a Gram (-) human pathogen, Helicobacter pylori, and consisted of 31 genes encoding the type-IV secretion system (Censini et al., 1996; Covacci et al., 1999; Covacci and Rappuoli 2000). It was suggested that Helicobacter pylori cag pathogenicity island (PAI) was inherited by horizontal transfer from unknown microorganisms (Covacci et al., 1999). It has recently been reported that type-IV secretion system exists in A. actinomycetemcomitans (Christie and Vogel, 2000; Covacci and Rappuoli, 2000). Although its precise roles for pathogenicity in A. actino7nycetemcomitans remain unclear, the potential functions of the type-IV secretion system per se in other species have been proposed (Asahi et al., 2000; Christie and Vogel, 2000; Covacci et al., 1999; Covacci and Rappuoli, 2000; Odenbreit et al., 2000; Stein et al., 2000). It was recently shown that cagE gene mutation in H. pylori resulted in a significant reduction of various gastric lesions in mongolian gerbils, suggesting that the type-IV secretion-encoded cagE gene is required for pathogenesis in vivo (Ogura et al., 2000). Further, Odenbreit et al. (2000) demonstrated that CagA, another protein encoded by the cag gene family, was translocated into the host epithelium by type-IV secretion machinery followed by being tyrosine-phosphorylated in situ (Asahi et al., 2000; Odenbreit et al., 2000; Stein et al., 2000). According to the present invention, but not to be bound to any particular theory, A. actinomycetemcomitans contains a putative type-IV secretion cag or cag-like gene family and that cagE is likely to be a member of the type-IV secretion system in A. actinomycetemcomitans. Furthermore, CagE can function as an effector protein by triggering apoptosis of human cells. Thus, it appears that the CagE protein of A. actinomycetemcomitans has much wider effects on human cells than its counterpart CagE in H. pylori (Covacci et al., 1999; Covacci and Rappuoli, 2000).

According to one embodiment of the present invention, the cagE gene or protein or a functional fragment thereof, can be used in a diagnostic screening method or in a diagnostic kit for the detection of the presence of A. actinomycetemcomitans. The diagnostic method and kit for the detection of A. actinomycetefncoffzztans can be used in detecting periodontal disease and other conditions or disease states where A. actinomycete7ncomitans has been found to be implicated in an increased risk of a systemic disorder. Examples of such conditions include thyroid and brain abscesses,

urinary tract infections, atherosclerotic plaque, sub-acute bacteria endocarditis, congestive heart diseases, stroke, pneumonia and pre-term low birth weight. In the context of the present invention a"functional fragment"includes a fragment of the cagE gene or protein that is also effective for use in a diagnostic screening method or in a diagnostic kit for the detection of the presence of A. actinomycetemcomitans.

Accordingly, the present invention provides a method of detecting A. actinomycetemcomitans or a condition associated with A. actinomycetemcomitans comprising assaying a sample for (a) a nucleic acid molecule encoding a CagE protein or a fragment thereof; (b) a CagE protein or a fragment thereof; or (c) an antibody that binds a CagE protein or a fragment thereof. In one embodiment, the condition associated with A. actinomycetemcomitans is a thyroid or brain abscess, a urinary tract infection, an atherosclerotic plaque, sub-acute bacteria endocarditis, congestive heart diseases, stroke, pneumonia and pre-term low birth weight.

(i) Nucleic acid molecules The nucleic acid molecules encoding CagE as described herein or fragments thereof, allow those skilled in the art to construct nucleotide probes for use in the detection of nucleotide sequences encoding CagE or fragments thereof in samples, preferably biological samples such as cells, tissues and bodily fluids. The probes can be useful in detecting the presence of a condition associated with A. actinomycetemcomitans. Accordingly, the present invention provides a method for detecting a nucleic acid molecule encoding CagE comprising contacting the sample with a nucleotide probe capable of hybridizing with the nucleic acid molecule to form a hybridization product, under conditions which permit the formation of the hybridization product, and assaying for the hybridization product.

Example of probes that may be used in the above method include fragments of the nucleic acid sequences shown in Figure 2C (SEQ ID NO: 1). A nucleotide probe may be labelled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as 32P, 3H, 14C or the like. Other detectable substances which may be used include antigens that are recognized by a specific labelled antibody, fluorescent compounds, enzymes, antibodies specific for a labelled antigen, and chemiluminescence. An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleic acid to be detected and the amount of nucleic acid available for hybridization. Labelled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed. ). The nucleotide probes may be used to detect genes, preferably in human cells, that hybridize to the nucleic acid molecule of the

present invention preferably, nucleic acid molecules which hybridize to the nucleic acid molecule of the invention under stringent hybridization conditions as described herein.

Nucleic acid molecules encoding a CagE protein can be selectively amplified in a sample using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic oligonucleotide primers from the nucleotide sequence shown in Figure 2C (SEQ ID NO: 1) for use in PCR. A nucleic acid can be amplified from cDNA or genomic DNA using oligonucleotide primers and standard PCR amplification techniques. The amplified nucleic acid can be cloned into an appropriate vector and characterized by DNA sequence analysis. cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al., Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc. , St. Petersburg, FL).

In another embodiment of the invention, the screening method involves a non-PCR based strategy. Such screening methods include two-step label amplification methodologies that are well known in the art. Both PCR and non-PCR based screening strategies can detect target sequences with a high level of sensitivity.

The most popular method used today is target amplification. Here the target nucleic acid sequence is amplified with polymerases. One particular preferred method using polymerase-driven amplification is the polymerase chain reaction (PCR).

The polymerase chain reaction and other polymerase-driven amplification assays can achieve over a million fold increase in copy number through the use of polymerase- driven amplification cycles. Once amplified, the resulting nucleic acid can be sequenced or used as a substrate for DNA probes.

When the probes are used to detect the presence of the target sequences, the biological sample to be analysed, such as blood, serum, dental plaque or saliva, may be treated, if desired to extract the nucleic acids. The sample nucleic acid may be prepared in various ways to facilitate detection of the target sequence; denaturation, restriction digestion, electrophoresis or dot blotting. The targeted region of the analyte nucleic acid usually must be at least single stranded to form hybrids with the target sequence of the probe. If the sequence is naturally single stranded, denaturation will not be required. However, if the sequence is double stranded, the sequence will need to be denatured. Denaturation can be carried out by various techniques known in the art, such as heating. Analyte nucleic acid and probes are incubated under conditions which permit stable hybrid formation of the target sequence in the probe with the putative target

sequence in the analyte. The region of the probe, which is used to bind to the analyte, can be made completely complementary to the target region. Therefore, high stringency conditions are desirable in order to prevent false positives. However, conditions of high stringency are used only if the probes are complementary to regions which are unique in the genome. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength of the hybridization or washing buffers, base composition, probe length and composition of formamide. These factors as well as methods of modifying these factors in order to obtain the desired stringency of hybridization are outlined in for example Maniatis et al. 1982 and Sambrook et al. 1989.

Detection of the resulting hybrid is usually accomplished by the use of labelled probes. Alternatively, the probe may be unlabelled, but may be detected by specific binding with a ligand which is labelled, either directly or indirectly. Suitable labels, and methods for labelling probes and ligands are known in the art and include, for example, radioactive labels which may be incorporated by known methods, biotin, flourescent groups, chemiluminescent groups, enzymes, antibodies and the like.

Variations of these basic schemes are known in the art and include those variations that facilitate separation of the hybrids to be detected from extraneous materials and/or that amplify the signal from the labelled moiety. A number of these variations are reviewed in Matthews & Kricka, 1988; Landegren et al. 1988 ; Mittlin, 1989; US Patent 4,868, 105 and EPO publication No. 225,807.

'As noted above, non-PCR based screening assays are also contemplated in this invention. According to this method, a nucleic acid probe is hybridized to the low level DNA target. This probe may have an enzyme covalently linked to the probe such that the covalent linkage does not interfere with the specificity of the hybridization. This enzyme-probe-conjugate-target nucleic constructs can then be isolated away from the free probe enzyme conjugate and a substrate is added for enzyme detection. These methods are also well known in the art (for example see Jablonski et al., 1986).

(ii) Proteins Besides the detection of cagE gene by use of a suitable nucleic acid probe, the corresponding protein or functional fragment thereof can be used as a diagnostic to screen for the presence of A. actinomycetemcomitans, specifically, antibodies produced as a result of A. actinomycetemcomitans infection. According to this aspect of the invention, the CagE protein, or functional fragment thereof, can be used in a method to detect antibodies in a sample from patients infected with A. actinomycetemcomitans. Accordingly, the present invention provides a method for detecting A. actinomycetemcomitans in a sample comprising contacting the sample with

a CagE protein which is capable of being detected after it becomes bound to an antibody to CagE in the sample. These methods include, for example, enzyme linked immunosorbent assay (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA). All of these detection methods are well known in the art (for example see US Patent 4,376, 110 or US Patent 4,486, 530). In this context a"functional fragment"includes a fragment of the CagE protein that can bind to antibodies found in a sample taken from a patient infected with A. actinomycetemcomitans.

In another embodiment, a sample from a patient may be assayed for the presence of CagE rather than antibodies to CagE. The CagE protein may be detected in a sample using antibodies that bind to the CagE protein which are described in detail in Section Ill (a). Accordingly, the present invention provides a method for detecting A. actinomycetemcomitans in a sample comprising contacting the sample with an antibody that binds to CagE which is capable of being detected after it becomes bound to the CagE in the sample.

Antibodies specifically reactive with CagE, or derivatives thereof, such as enzyme conjugates or labeled derivatives, may be used to detect CagE in various biological materials, for example they may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of CagE, and the antibodies. Examples of such assays are radioimmunoassays, enzyme immunoassays (e. g. ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination and histochemical tests. Thus, the antibodies may be used to detect and quantify CagE in a sample in order to determine its role in particular cellular events or pathological states, and to diagnose and treat such pathological states.

In particular, the antibodies of the invention may be used in immuno- histochemical analyses, for example, at the cellular and sub-subcellular level, to detect CagE, to localise it to particular cells and tissues and to specific subcellular locations, and to quantitate the level of expression.

Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect CagE. Generally, an antibody of the invention may be labelled with a detectable substance and CagE may be localised in tissue based upon the presence of the detectable substance. Examples of detectable substances include various enzymes, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, biotin, alkaline phosphatase, p-galactosidase, or acetylcholinesterase ; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or

phycoerythrin; an example of a luminescent material includes luminol ; and examples of suitable radioactive material include radioactive iodine I-125, I-131 or 3-H. Antibodies may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualised by electron microscopy.

Indirect methods may also be employed in which the primary antigen- antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against CagE. By way of example, if the antibody having specificity against CagE is a rabbit IgG antibody, the second antibody may be goat anti-rabbit gamma-globulin labelled with a detectable substance as described herein.

Where a radioactive label is used as a detectable substance, CagE may be localized by autoradiography. The results of autoradiography may be quantitated by determining the density of particles in the autoradiographs by various optical methods, or by counting the grains.

(d) CagE Modulators In addition to antibodies and antisense oligonucleotides described above, other substances that modulate CagE expression or activity may also be identified.

Accordingly, the present invention includes the use of the nucleic acids encoding CagE and the CagE protein to develop or identify substances that modulate CagE expression or activity. Such substances may be useful in the therapeutic applications described below.

(i) Substances that Bind CagE Substances that affect CagE activity can be identified based on their ability to bind to CagE.

Substances which can bind with the CagE of the invention may be identified by reacting the CagE with a substance which potentially binds to CagE, and assaying for complexes, for free substance, or for non-complexed CagE, or for activation of CagE. In particular, a yeast two hybrid assay system may be used to identify proteins which interact with CagE (Fields, S. and Song, O., 1989, Nature, 340: 245-247). Systems of analysis which also may be used include ELISA.

Accordingly, the invention provides a method of identifying substances which can bind with CagE, comprising the steps of : (a) reacting CagE and a test substance, under conditions which allow for formation of a complex between the CagE and the test substance, and (b) assaying for complexes of CagE and the test substance, for free substance or for non complexed CagE, wherein the presence of complexes indicates that the test substance is capable of binding CagE.

The CagE protein used in the assay may have the amino acid sequence shown in Figure 2C (SEQ ID NO: 2) or may be a fragment, analog, derivative, homolog or mimetic thereof as described herein.

Conditions which permit the formation of substance and CagE complexes may be selected having regard to factors such as the nature and amounts of the substance and the protein.

The substance-protein complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof. To facilitate the assay of the components, antibody against CagE or the substance, or labelled CagE, or a labelled substance may be utilized. The antibodies, proteins, or substances may be labelled with a detectable substance as described above.

CagE, or the substance used in the method of the invention may be insolubilized. For example, CagE or substance may be bound to a suitable carrier.

Examples of suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic Elm, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc.

The insolubilized protein or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.

The proteins or substance may also be expressed on the surface of a cell using the methods described herein.

The invention also contemplates assaying for an antagonist or agonist of the action of CagE.

(ii) Peptide Mimetics The present invention also includes peptide mimetics of the CagE protein of the invention. For example, a peptide derived from a binding domain of CagE will interact directly or indirectly with an associated molecule in such a way as to mimic the native binding domain. Such peptides may include competitive inhibitors, enhancers, peptide mimetics, and the like. All of these peptides as well as molecules substantially homologous, complementary or otherwise functionally or structurally equivalent to these peptides may be used for purposes of the present invention.

"Peptide mimetics"are structures which serve as substitutes for peptides in interactions between molecules (See Morgan et al (1989), Ann. Reports

Med. Chem. 24: 243-252 for a review). Peptide mimetics include synthetic structures which may or may not contain amino acids and/or peptide bonds but retain the structural and functional features of a peptide, or enhancer or inhibitor of the invention. Peptide mimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA 89: 9367); and peptide libraries containing peptides of a designed length representing all possible sequences of amino acids corresponding to a peptide of the invention.

Peptide mimetics may be designed based on information obtained by systematic replacement of L-amino acids by D-amino acids, replacement of side chains with groups having different electronic properties, and by systematic replacement of peptide bonds with amide bond replacements. Local conformational constraints can also be introduced to determine conformational requirements for activity of a candidate peptide mimetic. The mimetics may include isosteric amide bonds, or D-amino acids to stabilize or promote reverse turn conformations and to help stabilize the molecule.

Cyclic amino acid analogues may be used to constrain amino acid residues to particular conformational states. The mimetics can also include mimics of inhibitor peptide secondary structures. These structures can model the 3-dimensional orientation of amino acid residues into the known secondary conformations of proteins. Peptoids may also be used which are oligomers of N-substituted amino acids and can be used as motifs for the generation of chemically diverse libraries of novel molecules.

Peptides of the invention may also be used to identify lead compounds for drug development. The structure of the peptides described herein can be readily determined by a number of methods such as NMR and X-ray crystallography. A comparison of the structures of peptides similar in sequence, but differing in the biological activities they elicit in target molecules can provide information about the structure-activity relationship of the target. Information obtained from the examination of structure-activity relationships can be used to design either modified peptides, or other small molecules or lead compounds that can be tested for predicted properties as related to the target molecule. The activity of the lead compounds can be evaluated using assays similar to those described herein.

Information about structure-activity relationships may also be obtained from co-crystallization studies. In these studies, a peptide with a desired activity is crystallized in association with a target molecule, and the X-ray structure of the complex is determined. The structure can then be compared to the structure of the target molecule in its native state, and information from such a comparison may be used to design compounds expected to possess.

(iii) Drug Screening Methods

In accordance with one embodiment, the invention enables a method for screening candidate compounds for their ability to modulate (e. g. increase or decrease) the activity of a CagE protein. Such compounds may be useful in modulating apoptosis or cell adhesion or in treating conditions associated with A. actinomycetemcomitans.

The method comprises providing an assay system for assaying CagE activity, assaying the activity in the presence or absence of the candidate or test compound and determining whether the compound has increased or decreased CagE activity.

Accordingly, the present invention provides a method for identifying a compound that affects CagE protein activity or expression comprising: (a) incubating a test compound with a CagE protein or a nucleic acid encoding a CagE protein; and (b) determining an amount of CagE protein activity or expression and comparing with a control (i. e. in the absence of the test substance), wherein a change in the CagE protein activity or expression as compared to the control indicates that the test compound has an effect on CagE protein activity or expression.

In accordance with a further embodiment, the invention enables a method for screening candidate compounds for their ability to increase or decrease expression of a CagE protein. The method comprises putting a cell with a candidate compound, wherein the cell includes a regulatory region of a CagE gene operably joined to a reporter gene coding region, and detecting a change in expression of the reporter gene.

In one embodiment, the present invention provides culture systems in which cell lines which express the CagE gene, and thus CagE protein products, are incubated with candidate compounds to test their effects on CagE expression. Such culture systems can be used to identify compounds which upregulate or downregulate CagE expression or its function, through the interaction with other proteins.

In another embodiment, the present invention provides microchips that have nucleic acid molecules encoding CagE attached thereto. Such microchips are useful in high throughput screening assays for identifying drugs that interact with CagE.

Such compounds can be selected from protein compounds, chemicals and various drugs that are added to the culture medium. After a period of incubation in the presence of a selected test compound (s), the expression of CagE can be examined by quantifying the levels of CagE mRNA using standard Northern blotting procedure, as described in the examples included herein, to determine any changes in expression as a result of the test compound. Cell lines transfected with constructs expressing CagE can also be used to test the function of compounds developed to modify the protein expression. In addition, transformed cell lines expressing a normal CagE protein could be mutagenized by the use of mutagenizing agents to produce an altered phenotype in

which the role of mutated CagE can be studied in order to study structure/function relationships of the protein products and their physiological effects.

In the identification or design of potential drugs, the biologically active protein CagE, or fragment thereof, is used to identify small molecules with which they interact in order to fashion drugs which will interfere with the function of the polypeptide in vivo. In one approach, one first determines the three dimensional structure of the protein of interest or, for example, of the CagE receptor or ligand complex, by x-ray crystallography, by computer modelling, or most typically, by a combination of approaches. Less frequently, useful information regarding the structure of the polypeptide can be gained by modelling based on structure of homologous proteins. Thus, by one of these methods, one may design drugs which act as agonists or antagonists of CagE polypeptide activity. These inhibitors can be small molecules antibodies, monoclonal antibodies, or antibody fragments. Said inhibitors can therefore then be used to block apoptosis or to reduce bacterial cell adhesion or to treat A. actiiioinycetemconiitans mediated diseases.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of specifically binding the CagE polypeptide compete with a test compound for binding to the CagE polypeptide or fragment thereof. In this manner, the antibodies can be used to detect the presence of any polypeptide which shares one or more antigenic determinants of the CagE polypeptide. Antibodies can be prepared according to well known methods (Hurn and Chantler, 1980). A peptide, or a fragment thereof, is generally injected over a several month period into rabbits or other suitable animal. The sera is tested for immunoreactivity to the CagE polypeptide, or fragment thereof.

Once a potential drug or CagE modulator is identified using the above screening assays, it can be tested for therapeutic efficacy in a variety of in vitro or in vivo models. For example, whether or not a candidate compound is useful in modulating apoptosis or bacterial cell adhesion can be assessed using the methods described in the Examples.

(e) Therapeutic Uses As previously discussed, the CagE of the invention is involved in apoptosis, cell adhesion and conditions associated with A. actinomycetemcomitans.

Accordingly, the present invention includes the use of CagE and modulators thereof in therapeutic applications including the modulation of apoptosis and cell adhesion as well as in the treatment or prevention of conditions associated with A. actinomycetemcomitafas. The definitions used in this section apply to all embodiments of the invention unless otherwise noted.

In one embodiment, the present invention provides a method of modulating apoptosis comprising administering to a cell or animal in need thereof, an effective amount of agent that modulates CagE expression and/or activity. The invention also provides a use of an effective amount of an agent that modulates CagE expression and/or activity to modulate apoptosis or in the manufacture of a medicament to modulate apoptosis.

The term"agent that modulates CagE expression and/or activity"means any substance that can alter the expression and/or activity of CagE and includes agents that can inhibit CagE expression or activity (CagE antagonists) and agents that can enhance CagE expression or activity (CagE agonists). The agent can be any type of molecule including, but not limited to, proteins, peptides, nucleic acids (including DNA, RNA, antisense oligonucleotides, peptide nucleic acids), carbohydrates, organic compounds, small molecules, natural products and library extracts. Examples of agents which may be used to modulate CagE include nucleic acid molecules encoding CagE; the CagE protein as well as fragments, analogs, derivatives or homologs thereof ; antibodies; antisense nucleic acids; peptide mimetics; substances isolated using the screening methods described herein.

The term"modulate apoptosis"means that the agent causes an increase or decrease (i. e. change) in apoptosis as compared to the level observed in the absence of the agent.

The term"apoptosis"is intended to describe a cellular process of cell death characterized by the presence of cell shrinkage, nuclear collapse, and most typically DNA fragmentation.

The term"effective amount"as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired results.

The term"animal"as used herein includes all members of the animal kingdom, including humans.

Recent studies have shown that A. actinofnycetemcomitans is able to induce apoptosis of human epithelial cells, T cells, osteoblasts and macrophages in vitro (Kato et al., 1995; Kato et al., 2000; Kirby et al., 1995; Korostoff et al., 1998; Nalbant et al., 2000; Paju et al., 2000). Leukotoxin of A. actinomycetemcomitans can lyse human monocytes/macrophages by a pathway similar to apoptosis (Kato et al., 1995; Korostoff et al., 1998). However, Kato et al. (2000) showed that two leukotoxin- producing strains, ATCC 29523 and JP2 induced cytotoxicity on human KB cells just as Y4, a non-leukotoxin strain, did. This suggests that other factors were involved in the induction of apoptosis of human cells. Further, it has also been shown recently that A.

actinomycetemcomitans can penetrate oral epithelium via endocytosis-mediated transmigration for tissue invasion (Fives-Taylor et al., 1994; Meyer et al. ; 1996).

According to one aspect of the present invention, CagE of A. actinonaycetemcomitatas has been shown to be involved in inducing a relatively"early" apoptosis in various human cells, within 4-6 hrs. Interestingly, this process is independent of cytochalasin-mediated endocytosis, a process distinct from bacterial invasion-associated apoptosis which occurs relatively late (Fives-Taylor et al., 1994; Kato et al., 2000; Meyer et al. ; 1996). Not wanting to be bound to any particular theory, CagE is likely involved in a specific cascade of signaling events leading to apoptosis of host cells during A. actinomycetemcomitans infections.

In one embodiment, the present invention provides a method of inducing apoptosis comprising administering an effective amount of a CagE agonist to a cell or animal in need thereof. The present invention also provides a use of an effective amount of a CagE agonist to induce apoptosis or in the manufacture of a medicament to induce apoptosis.

The term"CagE agonist"includes any substance that can enhance the expression of a cagE gene and/or the activity of a CagE protein. Examples of CagE agonists include nucleic acid molecules encoding a CagE protein or fragments thereof and the CagE protein or fragments thereof. For inducing apoptosis, a CagE protein will preferably comprise amino acids 1-136 of the N-terminal end.

In another embodiment, the invention provides a method of inhibiting apoptosis comprising administering an effective amount of a CagE antagonist. The present invention also provides a use of an effective amount of a CagE antagonist to inhibit apoptosis or in the manufacture of a medicament to inhibit apoptosis.

The term"CagE antagonist"includes any substance that can inhibit the expression of a CagE gene and/or the activity of a CagE protein. Examples of CagE antagonists include antibodies and antisense molecules as described above.

The inventor has shown that CagE is involved in bacterial adherence. In particular, as shown in Example 5, a CagE mutant showed a significantly reduced ability to adhere to human epithelial cells demonstrating that CagE is involved in bacterial adherence in the host.

Accordingly, in another embodiment, the present invention provides a method of modulating cell adhesion comprising administering to a cell or animal in need thereof, an effective amount of agent that modulates CagE expression and/or activity.

The present invention also provides a use of an effective amount of an agent that modulates CagE expression and/or activity to modulate cell adhesion or in the manufacture of a medicament to modulate cell adhesion.

In one embodiment, the present invention provides a method of inducing cell adhesion comprising administering an effective amount of a CagE agonist to a cell or animal in need thereof. The present invention also includes a use of an effective amount of a CagE agonist to induce cell adhesion or in the manufacture of a medicament to induce cell adhesion.

In another embodiment, the present invention provides a method of preventing cell adhesion comprising administering an effective amount of a CagE antagonist to a cell or animal in need thereof. The invention also provides the use of an effective amount of a CagE antagonist to prevent cell adhesion or in the manufacture of a medicament to prevent cell adhesion.

The CagE antagonist is defined above and in this context can be any substance that will reduce or eliminate CagE's ability to act as a bacterial adhesion agent. This property is important for the treatment of numerous clinical conditions. In one example of this embodiment, the inhibitor can be used to treat periodontal infections in humans or other animals.

In a further embodiment, the present invention provides a method of modulating a condition associated with A. actinomycetemco71Zitans comprising of administering to a cell or animal in need thereof, an effective amount of agent that modulates CagE expression and/or activity. The present invention also provides a use of an effective amount of an agent that modulates CagE expression and/or activity to modulate a condition associated with A. actinomycetemcorzitans or in the manufacture of a medicament or modulating a condition associated with A. actiyaotnycetemcomitans.

In one embodiment, the present invention provides a method of preventing or treating a condition associated with A. actillonlycetemcolititans comprising administering to a cell or animal in need thereof an effective amount of a CagE antagonist. The present invention also provides a use of an effective amount of a CagE antagonist to treat or prevent a condition associate with A. actinomycetemcomitans or in the manufacture of a medicament for treating or preventing a condition associated with A. actinomycete7ncomitans.

In one embodiment, the CagE antagonist is an antibody to CagE as hereinabove described. In another embodiment, the CagE antagonist is an antisense oligonucleotide which is also described hereinabove.

The term"treatment or treating"as used herein means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i. e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease

progression, amelioration or palliation of the disease state, and remission (whether <BR> <BR> partial or total), whether detectable or undetectable. "Treating"can also mean prolonging survival as compared to expected survival if not receiving treatment.

The method may be used to treat any condition associated with A. actinomycetemcomitans including, but not limited to, periodontitis (including both of juvenile and adult forms); thyroid and brain abscesses, urinary tract infections, artherosclerotic plaque formation and sub-acute bacterial endocarditis. Since periodontitis is implicated in an increase risk for certain systemtic disorders such as congestive heart diseases, stroke, pneunomia, and pre-term low birth weight, methods of treating or preventing A. actinomycetemcomitans infection may also be used to treat these conditions.

(f) Pharmaceutical Compositions The above described substances including nucleic acids encoding CagE, CagE proteins, antibodies, and antisense oligonucleotides as well as other agents that modulate CagE may be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for administration in vivo. By "biologically compatible form suitable for administration iii vivo"is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects. The substances may be administered to living organisms including humans, and animals.

Administration of a therapeutically active amount of pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance to elicit a desired response in the individual. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

An active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc. ), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. If the active substance is a nucleic acid encoding CagE it may be delivered using techniques known in the art.

The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. , USA 1985) or Handbook of Pharmaceutical Additives (compiled by Michael and Irene Ash, Gower Publishing Limited, Aldershot, England (1995) ). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and may be contained in buffered solutions with a suitable pH and/or be iso-osmotic with physiological fluids. In this regard, reference can be made to U. S. Patent No. 5,843, 456. As will also be appreciated by those skilled, administration of substances described herein may be by an inactive viral carrier.

As hereinabove described, the present inventor has shown that CagE can induce apoptosis in cells. Accordingly, the present invention provides a pharmaceutical composition for modulating apoptosis comprising an effective amount of an agent that modulates CagE expression and/or activity in admixture with a suitable diluent or carrier. The present invention also provides a pharmaceutical composition for preventing apoptosis comprising an effective amount of a CagE antagonist in admixture of a suitable diluent or carrier. The present invention further provides a pharmaceutical composition for inducing apoptosis comprising an effective amount of a CagE agonist in admixture with a suitable diluent or carrier.

In a preferred embodiment, a CagE protein or functional fragment thereof can be used as an active ingredient in a pharmaceutical composition for inducing apoptosis. In one example of this aspect of the invention CagE protein or functional fragment thereof can be used to induce apoptosis for the treatment of cancer. As shown in the examples, CagE polypeptide was effective in inducing apoptosis in KB epithelial cells. Thus, according to this aspect of the invention, there is provided a pharmaceutical formulation comprising an effective amount of CagE protein or a fragment thereof, which is effective in inducing apoptosis in human cells. The optimum pharmaceutical formulation of the protein or fragment thereof can be determined by one skilled in the art depending upon the route of administrative and desired dosage. The CagE protein or fragment thereof can be produced by expression of the corresponding nucleic acid sequence in bacteria, for example, using known expression vectors. In addition, the techniques of synthetic chemistry can be used to synthesise either the CagE protein or suitable fragment thereof. Active CagE molecules can be introduced into the cells by

microinjection or by use of liposoms, for example. One could target CagE protein to cancer cells via surface markers and induce apoptosis. Alternatively, some active molecules may be taken up by the cells actively or by diffusion. Extracellular application of the CagE protein, or fragment thereof, may be sufficient to induce apoptosis in tumour cells. Alternatively, the CagE protein or effective fragment thereof may be fused to a carrier molecule to ensure proper delivery of the protein or fragment to the cancer site. Suitable compounds which could be used to target the CagE protein or fragment thereof to the cancer cells are known in the art.

According to a further aspect of this invention, the CagE protein or functional fragment thereof, can be used as an active ingredient in a pharmaceutical composition to promote bacterial cell adhesion. Accordingly, the present invention provides a pharmaceutical composition for promoting bacterial cell adhesion comprising an effective amount of a CagE agonist in admixture with a suitable diluent or carrier.

Alternatively, an inhibitor of the CagE protein, as described above, can be used as an active ingredient in a pharmaceutical composition to inhibit bacterial cell adhesion.

Accordingly, the present invention provides a pharmaceutical composition for inhibiting bacterial cell adhesion comprising an effective amount of a CagE antagonist in admixture with a suitable diluent or carrier. It is widely believed that bacterial cell adhesions is a necessary step which is required for bacterial infection. Thus, blocking this step can have important clinical implications for not only treating periodontal disease, but other bacterial diseases as well.

(g) Vaccines The present invention includes vaccines for treating or preventing A. actiyzomycetemcomitans mediated diseases.

In one embodiment, the vaccine comprises a CagE protein in admixture with a suitable diluent or carrier. In another embodiment the vaccine comprises a nucleic acid encoding a CagE protein in admixture with a suitable diluent or carrier. The term "CagE protein"is as previously defined herein to include all homologs, analogs, fragments, or derivatives of the CagE protein from A. actinomycetemcomitans. The fragments of the CagE protein will be sufficient in order to elicit an immune response against A. actinomycetemcomitans.

In a further embodiment, the vaccine comprises an antibody that binds to a CagE protein in admixture with a suitable diluent or carrier. Such antibodies can be prepared as hereinbefore described.

The vaccine can either include a protein (such as a CagE protein or antibody that binds a CagE protein or peptide) or as a nucleic acid encoding the CagE protein. Such nucleic acids include free or naked RNA or DNA or in a vector. In a

preferred embodiment, the nucleic acid sequence is contained in a vector or plasmid. In one embodiment, the vector may be viral such as poxvirus, adenovirus or alphavirus.

Preferably the viral vector is incapable of integration in recipient animal cells. The elements for expression from said vector may include a promoter suitable for expression in recipient animal cells.

The vaccines of the invention may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like to form suitable vaccine formuations. The vaccines can also be lyophilized. The vaccines may also contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. In this regard, reference can be made to U. S. Patent No. 5,843, 456. Reference can also be made to the textbook Vaccine Design: the Subunit and Adjuvant Approach, Michael F. Powell and Mark J. Newman, eds. Plenum Press, New York, 1995.

The vaccine may additionally include adjuvants to enhance the immune response to the CagE protein or antibody. A wide range of extrinsic adjuvants can provoke potent immune responses to antigens. These include saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria and mineral oil, Freund's complete adjuvant, bacterial products such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes.

U. S. Patent No. 4,855, 283 granted to Lockhoff et al on August 8,1989, which is incorporated herein by reference thereto, teaches glycolipid analogues including N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which is substituted in the sugar residue by an amino acid, as immuno-modulators or adjuvants.

Thus, Lockhoff et al. (Chem. Int. Ed. Engl. 30: 1611-1620 (1991) ) reported that N- glycolipid analogs displaying structural similarities to the naturally-occurring glycolipids, such as glycophospholipids and glycoglycerolipids, are capable of eliciting strong immune responses in both herpes simplex virus vaccine and pseudorabies virus vaccine. Some glycolipids have been synthesized (from long chain-alkylamines and fatty acids that are linked directly with the sugars through the anomeric carbon atom) to mimic the functions of the naturally occurring lipid residues.

U. S. Patent No. 4,258, 029 granted to Moloney and incorporated herein by reference thereto, teaches that octadecyl tyrosine hydrochloride (OTH) functions as an adjuvant when complexed with tetanus toxoid and formalin inactivated type I, II and in poliomyelitis virus vaccine. Nixon-George et al. (J. Immunol. 14: 4798-4802 (1990)) have also reported that octadecyl esters of aromatic amino acids complexed with a

recombinant hepatitis B surface antigen enhanced the host immune responses against hepatitis B virus.

Adjuvant compounds may also be chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative.

Adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross- linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Preferably, a solution of adjuvant according to the invention, especially of carbomer, is prepared in distilled water, preferably in the presence of sodium chloride, the solution obtained being at acidic pH. This stock solution is diluted by adding it to the desired quantity (for obtaining the desired final concentration), or a substantial part thereof, of water charged with NaCl, preferably physiological saline (NaCL 9 g/1) all at once in several portions with concomitant or subsequent neutralization (pH 7.3 to 7.4), preferably with NaOH.

This solution at physiological pH will be used as it is for mixing with the vaccine, which may be especially stored in freeze-dried, liquid or frozen form. The polymer concentration in the final vaccine composition will be 0. 01% to 2% w/v, more particularly 0.06 to 1% w/v, preferably 0.1 to 0.6% w/v.

Persons skilled in the art can also refer to U. S. Patent No. 2,909, 462 (incorporated herein by reference) which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups (preferably not more than 8), the hydrogen atoms of the at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms (e. g. vinyls, allyls and other ethylenically unsaturated groups). The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with allyl sucrose or with allyl pentaerythritol. Among them, there may be mentioned Carbopol (for example, 974P, 934P and 971P). Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers EMA (Monsanto; which are copolymers of maleic anhydride and ethylene, linear or cross-linked, (for example cross-linked with divinyl <BR> <BR> ether) ) are preferred. Reference may be made to J. Fields et al. (Nature, 1960,186 : 778- 780) for a further description of these chemicals (incorporated (herein by reference).

Adjuvants for parenteral immunization include aluminum compounds (such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate). The antigen can be precipitated with, or adsorbed onto, the aluminum compound according to standard protocols. Other adjuvants such as RIBI (ImmunoChem, Hamilton, MT) can also be used in parenteral administration.

Adjuvants for mucosal immunization include bacterial toxins (e. g. ,<BR> cholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridium difficile toxin A and the pertussis toxin (PT), or combinations, subunits, toxoids, or mutants thereof).

For example, a purified preparation of native cholera toxin subunit B (CTB) can be of use. Fragments, homologs, derivatives, and fusion to any of these toxins are also suitable, provided that they retain adjuvant activity. Preferably, a mutant having reduced <BR> <BR> toxicity is used. Suitable mutants have been described (e. g. , in WO 95/17211 (Arg-7- Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9- <BR> <BR> Lys and Glu-129-Gly PT mutant) ). Additional LT mutants that can be used in the methods and compositions of the invention include, for example Ser-63-Lys, Ala-69- Gly, Glu-110-Asp, and Glu-112-Asp mutants. Other adjuvants (such as a bacterial monophosphoryl lipid A (MPLA) of various sources (e. g. , E. E.coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri, saponins, or polylactide glycolide (PLGA) microspheres) can also be used in mucosal administration.

Adjuvants useful for both mucosal and parenteral immunization include polyphosphazene (for example, WO 95/2415), DC-chol (3 b- (N- (N', N'-dimethyl aminomethane)-carbamoyl) cholesterol (for example, U. S. Patent No. 5,283, 185 and WO 96/14831) and QS-21 (for example, WO 88/9336).

Accordingly, the present invention provides a method of eliciting an immune response to A. actinomycetemcomitans comprising administering an effective amount of a vaccine formulation comprising a CagE protein, a nucleic acid encoding a CagE protein or an antibody to a CagE protein to an animal in need thereof.

The term"eliciting an immune response"is defined as causing, enhancing, or improving any response of the immune system, for example, of either a humoral or cell-mediated nature. Whether a vaccine or antigen elicits an immune response can be assessed using assays known to those skilled in the art including, but not limited to, antibody assays (for example ELISA assays), antigen specific cytotoxicity assays and the production of cytokines (for example ELISPOT assays).

The term"an effective amount"of the vaccine of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result (e. g. elicit an immune response to A. actinomycete77lcomitans). The effective amount of a compound of the invention may vary according to factors such as the disease state, age, sex, and weight of the animal. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigences of the therapeutic situation.

The term"animal"as used herein includes all members of the animal kingdom, including humans. Preferably, the animal to be treated is a human.

The term"administering"is defined as any conventional route for administering an antigen to an animal for use in the vaccine field as is known to one skilled in the art. This may include, for example, administration via the topical, oral and parenteral (i. e. subcutaneous, intradermal, intramuscular, etc. ) routes and further includes, transdermal and mucosal delivery, including mucosal delivery accomplished by oral feeding, inhaling and through the membranes accessible through the terminal portions of the large intestine.

A particularly preferred method of immunizing an animal with the vaccine encompasses a prime-boost protocol. Typically, a prime-boost protocol involves an initial administration of the vaccine followed by a boost of the vaccine. This protocol will elicit an enhanced immune response relative to the response observed following only one administration of the vaccine. An example of a prime-boost methodology/protocol is described in WO 98/58956, which is incorporated herein by reference. In the prime-boost protocol, the route of administration for the priming does not have to be the same route as used for the boosting.

The vaccine formulation may be administered with other agents including other adjuvants as well as immune stimulatory molecules including cytokines.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples.

These examples are described solely for the purpose of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

EXAMPLES EXAMPLE 1: Cloning, characterization and expression of the cagE gene, a type-IV secretion protein in A. actinomycetemcomitans Clinical patients Four localized juvenile periodontitis subjects (called JP1-JP4; 3 M & 1 F; mean age: 21 4.4 years), one adult periodontitis subject (called AP; M; age of 27) who was exposed to A. actinomycetemcomitans infection and one healthy control subject (called HC; M; age of 26) without clinical evidence of periodontal disease were recruited for blood donation as described previously (Teng et al., 1999; Teng et al., 2000). Informed consent was obtained from the patients and all protocols used in this

study were approved by the Human Ethics and Animal Experimentation Committees of the University of Western Ontario (UWO), London, Ontario.

Mammalian tissue culture and bacterial growth conditions All mammalian tissue cultures including human epithelial KB cells and lymphoblastic Jurkat T cell lines were maintained in complete RPMI-1640 medium supplemented with 10% FBS (Gibco, ON) at 37°C under 5% CO incubation. Human primary epithelial cells prepared from gingival discards during periodontal surgeries at UWO dental clinic were grown and maintained in Epilife medium supplemented with pituitary extracts (Sigma Canada, ON) according to the manufacturer's protocol.

Human primary osteoblastic cells were purchased from Clonetics (MD, USA) and grown and maintained in complete OBM basal medium supplemented with 10% FCS.

Human primary T cells were grown and maintained in complete RPMI-1640 medium supplemented with 10% FCS plus 2-5% donor's serum. Human primary endothelial cells from lung micro-vasculature were purchased from Clonetics and were grown and maintained in EGM-2MV basal medium supplemented with 10% FCS according to manufacturer's protocol.

A. actinomycetemcomitans strains JP2 (ATCC-29523), Y4 (ATCC- 43781), UP49, UP53 and HK1651 were grown in an anaerobic chamber (Coy, MI; CO2, 10% H2 and 85% N2) with TSBY broth, consisting of a Tripticase soy broth supplemented with 0.6% (wt/vol) yeast extract and 0.04% (wt/vol) NaHCO3 of pH 7.2 at 37°C for 24-48 hrs. E. coli strains DH5a (Gibco, ON) and SG13009 (Qiagen, QC) were used as hosts for recombinant plasmids. E. coli cultures were grown with shaking at 250 rpm under 37°C in LB broth containing 50 Fg/ml of ampicillin if needed.

Screening of A. actiizomyceteincomitans genomic library for bacterial antigens recognized by human CD4+ T cells Genomic DNA of A. actinomycetemcomitans was isolated according to the methods described previously (Sambrook et al. 1989), then partially digested by Sau3Al (Gibco, ON) and size-fractionated via sucrose gradient (8-50%) centrifugation.

Pooled DNA of size 0.5-4. 0 kb was collected, purified and then ligated into the Bgl-H site of pTrcHis-C vector (Invitrogen, CA) after being dephosphorylated with calf intestinal alkaline phosphatase (New England Bio-Lab, MA, USA). Ligated plasmids were transformed into E. coli Top 10 strain (Invitrogen, CA) by eletroporation, and then plated onto selective broth to quantify transformation. Further restriction enzyme digestion of plasmid DNA of E. coli transformants showed that the majority (>90%) of the yielded clones carried a new DNA insert (data not shown). The size of the genomic library used for screening was estimated to be around 106clones/llg DNA insert.

Periodontal CD4+ T cells were purified from Aa-inoculated HuPBL-NOD/SCID mice

as described previously (Teng et al., 1999) and then expanded by rhIL-2 for 10-14 days in vitro. Later, these CD4+ T cells were transfected with linearized DNA vector prepared from an IL-2-inducible reporter construct with a lacZ sequence (cx-12 : NF-AT-lacZ, kindly provided by Dr. G. Crebtree, Stanford University, CA) by a Gene-Pulser II (Bio- Rad, CA) as described elsewhere (Fiering et al., 1990; Sanderson et al., 1995).

Hygromycin-resistant T cells were then steadily selected and expanded in hrIL-2. Thus, T cell activation can be measured and visualized as lacZ expression in single cells or in bulk cultures, as described previously (Fiering et al., 1990; Sanderson et al., 1995) after in vitro stimulation. Controls included the sham-or/and lacZ-only transfected CD4+ T cells, which did not yield any positive clones from the screening described below.

To screen bacterial antigens recognized by human CD4+ T cells, individual E. coli from a genomic DNA library of A. actiytomycetemcomitans were grown in pools (30-50 bacterial colonies) in selective LB medium containing 50ug/ml of ampicillin, then plated into 96-well microplates overnight and replicate cultures were made by splitting. IPTG was added in vitro to induce protein expression in the last 4 hrs. In parallel, patients'HuPBL-derived monocytes/macrophages (1-2. 5xl05/well) were prepared as described (Sanderson et al., 1995) and cultured overnight with rhIFN- y (lOOug/ml) to up-regulate HLA expression. Then replica bacteria in 96-plates were co- cultured with IFN-y-activated macrophages/monocytes for 1-2 hrs to allow phagocytosis of bacteria, which was followed by adding gentamycin (100pg/ml) in order to kill and remove bacteria in the culture supernatants. Meanwhile, periodontal CD4+ T cells from Aa-inoculated HuPBL-NOD/SCID mice (the same donor) after being transfected with NF-AT-lacZ described above, were added to the co-cultures at 3-5xlO4cells/well for overnight incubation. The lacZ (+) cells were visualized by staining with buffered X-gal (lmg/ml) as described elsewhere (Fiering et al., 1990; Sanderson et al., 1995). The positive wells giving blue signals were sequentially subjected to further screening for confirmation by using fewer clones (i. e. , 5-10) per well, until a single positive clone was identified. In the current study, the positive DNA clones identified were experimentally confirmed for at least 2-3 times by using 2 to 4 different patients'samples tested in vitro. In addition, to avoid non-specific recognition of E. coli antigens in the co-culture experiments during the screening, hyper-immune mouse serum against E. coli (TOP-10) strain was raised in Balb/c mice, then papain-treated to generate Fab'fragments to avoid Fc receptor-mediated endocytosis or/and activation of monocytes/macrophages in vitro.

A typical picture of positive clones in bulk culture mixed with A. actiyzomycetemconzitans-activated periodontal CD4+ T cells showing blue staining is shown in Fig. 1A, whereas the control CD4+ T cells carrying cx-12 vector alone without

NFAT-IL-2 and lacZ sequences show a background pale color without blue staining in vitro in Fig. 1B.

Cloning, expression andpurification of the recombinant CagEprOteiyz After screening the genomic DNA library of A. actinomycetemcomitans through expression cloning as described above, several novel DNA sequences were obtained, which were then subjected to worldwide search of the GenBank for sequence homology and comparison. One clone identified (F023) contained a partial DNA sequence with significant homology to cagE gene of Helicobacterpylori (Censini et al., 1996; Tummuru et al., 1995). By searching A. actinomycetemcomitans sequences available at the University of Oklahoma (www. genome. ou. edu/act. html), an open reading frame (ORF) encoding a protein of 340 residues, with a predicted MW of 38.6 kDa was identified. The ORF of cagE gene without the first ATG start codon was amplified by PCR using the following PCR primers: 5'- GGATCCGTCCCTGAAATTTTATTAGCTTG-3' (forward primer with a BamHI site at the 5'-end, underlined (SEQ ID NO: 11) ) and 5'-CTGCAG TTAAACGACCTTTAAACATTTTTTTA-3' (reverse primer with a PstI site at the 5'- end, underlined (SEQ ID NO: 12) ). The PCR program employed was as follows: preheated at 94°C for 3 min ; followed by 94°C for 1 min, 56°C for 1 min, 72°C for 1 min, of 35 cycles, 72°C for 10 min. Then, the cagE gene ORF was cloned into the BamHI and PstI sites of pQE30 vector with 6-Histidine tag (Qiagen, ON), resulting in the recombinant pQE30-cagE in which the recombinant protein contained a 6xHis tag followed by residues encoded by the Aa-cagE ORF. Recombinant vector pQE30-cagE was then introduced into E. coli SG13009 strain by electroporation for protein expression. Western blot analysis showed that the recombinant CagE protein had a MW of 39 kDa (Fig. 2A), which is consistent with the predicted molecular weight. The entire DNA coding sequence and predicted protein sequence of the cagE gene in various A. actinomycetemcomitans strains were found to be identical (i. e. , JP2, Y4, and HK1651) and were subsequently deposited into the GenBank (GenBank accession numbers: AF319466, AF319465 and AF319467, respectively).

The deduced amino acid sequence of A. actinornycetemcomitans CagE protein showed significant homology (Fig. 2B) to those of H. pylori CagE and Agrobacterium tumefaciens (A. tumefaciens) VirB4, both of which are encoded by a type-IV secretion system associated with virulence or pathogenesis in their respective hosts (Censini et al., 1996; Tummuru et al., 1995; Covacci et al., 1999; Chrisitie and Vogel, 2000; Covacci and Rappuoli, 2000). The CagE protein of A. actinomycetemconiitayas is shorter (only 340 a. a), than its counterparts H. pylori CagE (981 a. a) and A. tumefaciens VirB4 (789 a. a). Sequence alignment showed that CagE

protein in A. actinofnycetemcomitans shared high homology to the C-termini (after residue 300) of its counterparts (Fig. 2B). However, in this homologous region, there is neither comparable conserved NTPase sequences nor nucleotide-binding Walker A &B motifs present. Specifically, a. a. residues of A. actinomycetemcomitans CagE were 29% identical to those of H. pylori CagE and 21% to A. tumefaciens VirB4. More specifically, the C-terminal residues of A. actinomycetemcomitans CagE (within 137 and 340 amino acids residues) were 39% identical to those of H. pylori CagE and 27% to A. tumefaciens VirB4. In addition, A. actinomycetemcomitans CagE also showed significant homology (about 20% identical) to VirB4 proteins of Bordetella pertussis, Bartonella henselae and Brucella abortus, an invasion-associated protein (Opc) from Neisseria meningitidis, and TraB protein of E. coli (review: Chrisitie and Vogel, 2000; data not shown).

To further investigate whether there are any putative cag or cag-like type- IV secretion gene family (i. e., cag Pathogenecity Island-PAI) in A. actinoniycetemcomitans, genomic DNA fragment (of HK 1651 strain) flanking the cagE gene was cloned and analyzed by genomic DNA walking and sequencing. The results showed that, within ~6 kb flanking region around the cagE gene, there are two more putative ORFs showing homology to the cagD and cagH genes of H. pylori (Censini et al., 1996; data not shown). More specifically, the cagD (or cagD-like) homologue (25% identity) is located (-3kb) at the 5'-end of the cagE gene while the cagH (or cagH-like) homologue (19% identity) is located (-lkb) at the 3'-end of the cagE gene. However, the N-terminal region of the cagE gene is distinctly different in its sequences and functions. In addition, our latest search of the A. actinomycetemcomitans genomic sequence database (www. genome. ou. edu/act. html) revealed a few more putative ORFs carrying homology to the bacterial type-IV secretion proteins, including CagC and CagM of H. pylori ; VirB 1 and VirB 11 of A. tumefaciens (sequence alignment not shown). Therefore, it appears that there is a putative type-IV secretion cag or cag-like PAI in A. actinomycetemcomitans and that the cagE gene the inventor reports here is a member of the type-IV secretion family genes.

Interestingly, the cagE gene appeared to exist among all A. actinofraycetemcomitans strains tested, including JP2, Y4, HK1651, UP49 and UP53 as assessed by PCR amplification of the 1 kb DNA fragment using the same PCR primer set (Fig. 2C). Further DNA sequencing study confirmed that the nucleotide sequences of the cagE gene from various A. actinomycetemcomitans strains tested above shared >99% homology (data not shown). Thus, these data suggest that the cagE gene is highly conserved among A. actinomycetemcomitans strains.

Expression and purification of recombinant CagE fusion protein was performed according to the manufacturer's protocols. Briefly, SG13009 cells were grown and monitored until they reached 0.5 at OD600, then were subjected to IPTG induction for 4 hrs after which cells were pelleted and lysed in buffers. Then protein was purified on a Ni-NTA agarose column according to the manufacturer's instructions (Qiagen, ON). Purified protein was subjected to a 12.5 % SDS-PAGE analysis and the 39-kDa CagE protein band was excised after brief staining with Coomassie-Blue. The CagE protein was then eluted with 50mM ammonium bicarbonate containing 0. 01% SDS (pH 7.4) and further dialysed in PBS for 48 hrs. Finally, purified protein was re- suspended in 50 mM PBS (pH 7.0) and stored at-80°C. Protein purity was further assessed by SDS-PAGE with Coomassie-Blue and Silver staining in which only one detectable protein band of 39 kDa representing CagE was observed (data not shown).

EXAMPLE 2: Expression of CagE protein is biologically and clinically significant Immunoblot analysis and ELISA A polyclonal Ab serum against CagE protein was raised in five Balb/c mice by intra-peritoneal immunization of 50 ug purified protein in 100 pl CFA per mouse at day 1, followed by 2-3 i. p. boosting immunizations using the same amounts of protein in IFA at three-week intervals. Ten days after the last boost, cell-free serum samples were collected by centrifugation (10,000 rpm, 10 min at 4°C) and then pooled for studies of serum titers and Ab specificity by ELISA and Western blotting. To analyze native CagE protein in bacteria, individual A. actinomycetemcomitans strains were grown in an anaerobic chamber at conditions described above for 24 hrs. Bacterial lysate and supernatants were prepared by centrifugation (5000g, 5 min) and supernatants were further concentrated 50x by using Centricon Plus-20 according to the manufacturer's instructions (Millipore, MA). These samples were subjected to SDS- PAGE and transferred to nitrocellulose filters. The filters were blocked with PBS containing 5% skim milk for 2 hrs at room temperature and then incubated with polyclonal mouse sera raised above (1: 500 diluted in PBS with 0.5% BSA) at 4°C overnight. After washing 3x with PBS containing 0.25% Tween-20, filters were incubated with goat anti-mouse IgG alkaline phosphatase (AP)-conjugate for 1 h at room temperature, washed 3x with PBS (in 0.25% Triton) and finally detected with AP substrate. ELISA was performed according to the method described previously (Teng et al., 1999) by using either diluted patients'serum or the polyconal Ab mouse sera specific to CagE as described above.

The polyclonal Ab sera raised against recombinant CagE protein specifically recognized the 39 kDa recombinant CagE protein expressed in E. coli (Fig.

3A). To study the native form of CagE protein produced by the bacterium, different A. actinomycetemcomitans strains were grown and analyzed by Western blot using the polyclonal CagE Ab. The results showed that the 39-kDa CagE protein was detected in both the cell lysate and supernatant (SN) of Y4, JP2 and HK1651 strains, consistent with the predicted molecular weight (Fig. 3B). These results suggest that expression of the CagE protein existed among various A. actinomycetemcomitans strains that contain a bacterial type-IV secretion system described recently (Christie and Vogel, 2000; Covacci and Rappuoli, 2000). Despite slightly more CagE proteins were detected in the culture supernatant than in the cell lysate (Fig. 3B), it remains to be determined whether the CagE proteins detected in the cultural supernatant were indeed a secreted protein or a structural component that sheds, or even both.

To study the clinical significance of CagE protein expressed by A. actinontycetemcomitans, serum samples from five periodontitis (JP1-JP4 and AP) subjects associated with A. actinomycetemcomtans infection and a clinically healthy control (HC) subject were analyzed by ELISA. The results showed that sera from all of the five patients recognized the purified CagE protein with significantly higher IgG titers than that from the HC sample (Fig. 4A). Consistently, sera from the above periodontitis patients readily recognized the 39-kDa CagE protein in Western blot analysis (Fig. 4B).

Further, our recent studies also showed that CagE protein induced immune recognition by LJP-derived human T cells and mouse T cells in an in vitro proliferation assay (data not shown). Thus, these data strongly suggest that CagE protein expressed by A. actinomycetemcomitans was clinically relevant as recognized by human immune cells and was associated with the disease process.

EXAMPLE 3: CagE protein induces apoptosis of human cells Cytotoxicity assay Some bacterial type-E secretion proteins can function as an effector by inducing apoptosis of the host cells (Mills et al., 1997, Chen et al., 1996), however, to date, it has not been shown that any bacterial type-IV secretion protein (s) is functionally manifested in a similar fashion (Christie and Vogel, 2000). Thus, the inventor investigated whether CagE, a type-IV secretion protein in A. actinomycetemcomitans may function as an effector via inducing apoptosis of human cells. KB cells (4 x 103 cells/well) were seeded in a 96-well flat-bottom culture plate and incubated at 37°C in a 5% CO2 atmosphere in the presence of CagE at indicated concentrations or a control protein, OVA, chicken ovalbumin (Sigma Canada, ON). Cell viability was assessed after 24 hrs with the addition of 10 p1 WST-1 (Roche Inc. , QC), which measures the mitochondrial dehydrogenase activities in viable cells via the cleavage of the tetrazolium salt WST-1 according to the manufacturer's protocols. Plates were analyzed using a

Bio-Rad ELISA reader at 405nm. Then, the percentage cytotoxicity was calculated by the following formula: 100 x [(OD40s in untreated control cells-OD40s in treated cells)/ OD40s in untreated control cells]. The results showed that CagE protein at 0.1-0. 5 pm concentrations induced significant apoptosis of human primary epithelial cells within 6- 12 hrs (Fig. 5A, top-right panel), whereas no significant apoptosis was observed in co- cultures treated with OVA or medium-only controls (Fig. 5A, top-left panel). After 6 to 12 hrs, CagE treated human epithelial cells showed morphological characteristics of apoptosis such as membrane blebbing and nuclear and cellular condensations in majority (>80%) of the cell population (Kato et al., 2000). Similar results were observed in CagE-treated human epithelial KB cell line (Fig. 5A). Similar results were obtained with other cell types in the periodontium, human primary endothelial cells, osteoblasts and T cells were co-cultured with purified CagE protein (Fig. 5A).

Analysis of host DNA fragmentation post bacterial infection in vitro Analysis of DNA fragmentation was performed as described elsewhere <BR> <BR> (Sambrook et al., 1989). Briefly, 104 mammalian cells (i. e. , human primary cells, KB cells or Jurkat cell line) per well were incubated at 37°C in 24-well plates from 4-6 hrs up to 24 or 48 hrs in the presence or absence of purified CagE protein at indicated concentrations or appropriate ratios (human cell: bacteria = 1: 500 to 1: 500,000) of A. actinomycetemcomitans. To assess the killing of mammalian cells induced by A. actinofnycetemcomitans in vitro, when needed, bacteria were added into the tissue cultures at 37°C in 5% CO2 incubator for up to 4-6 hrs. To maintain bacterial viability for infecting the mammalian cells in co-cultures, fresh culture medium and bacteria of the same concentrations were replaced at a 3-hr-interval before the end of the experiment. Then, at the indicated time point, the mammalian cells were harvested after washing 3x with medium and PBS to remove extra cellular bacteria and then re- suspended in 200ut of TE buffer. The resulting genomic DNA was isolated and purified as described and then assayed in 1% agarose DNA gel for electrophoresis (Sambrook et al., 1989, Buommino et al. 1999). The results showed DNA ladder formation in CagE-treated KB epithelial cells after 6 hrs, but not in OVA-or PBS- treated control or untreated cells (Fig. SB), suggesting an inter-nucleosomal cleavage of the cells undergoing apoptosis. Similar results were seen with other cell types in the periodontium, human primary endothelial cells, osteoblasts and T cells were co-cultured with purified CagE protein (Fig. 5B).

TdT-fnediated deoxyuridine 5'-tyzphosphate nick end labeling (TUNEL) assay The study of DNA fragmentation was performed by using an Apoptosis Detection Kit System which provides the terminal deoxytransferase-mediated dUTP nick end labeling of DNA strand breaks in green fluorescence according to the

manufacturer's protocol (Promega, CA). The results of a TUNEL study confirmed that apoptosis was, indeed, induced by CagE protein at the single cell level (see below in Fig.

6C). Furthermore, the results of cytotoxicity assay also showed that purified CagE protein significantly decreased the viability of human epithelial cells after co-culturing in vitro (Fig. 5C). Thus, it is evident that purified CagE protein at micro-molar concentrations induced significant apoptosis of various human cells in vitro.

EXAMPLE 4: The cagE mutant fails to induce apoptosis of human epithelial cells Generation of cagE-mutant To generate a cagE integration mutant in A. actinomycetemcomitans, an internal DNA fragment of the cagE gene encoding a. a. residues 38 to 221 (of 340 a. a.) was amplified by PCR using the following primers: forward primer 5'- GGATCCGATATTCCTCTTGCTCAACAGC-3' (with a BamHI site at the 5'-end, <BR> <BR> underlined (SEQ ID NO: 13) ), and reverse primer 5'-CTG CAGCCATTCGGCGACCATCAAGC-3' (with a PstI site at the 5'-end, underlined <BR> <BR> (SEQ ID NO: 14) ). The PCR transcript was then cloned into the BamHI and PstI sites of pUS 19, a ColEl-based vector carrying spectinomycin and ampicillin resistance genes (vector kindly provided by Dr. W. Haldenwang at the University of Texas, Health Science Centre at San Antonio;) to generate recombinant pUS19-cagE. The pUS19- cagE was purified and then electroporated into A. actinomycetemcomitans (JP2 strain) and resistant colonies were selected as previously described (Kolodrubetz et al., 1995).

Stable integrated cagE mutants were isolated and confirmed by subsequent PCR analysis and Southern blotting (data not shown).

CagE protein was neither detected in the culture supernatant (SN) nor in the cell lysate of the cagE mutant (Fig. 6A). Subsequently, the cagE mutant was used to test its ability to induce apoptosis on human KB epithelial cells for in vitro infectivity as described in Experimental Procedures. As shown in Fig. 6B, 4 hr post-treatment, WT JP2 strain induced DNA fragmentation on KB cells, whereas cagE mutant failed to induce DNA ladder formation. To detect the apoptosis observed above at single cell level, a TUNEL assay was employed. The results showed that significantly more TUNEL-positive cells were detected in WT-treated KB cells than in cagE mutant- treated KB cells (Fig. 6C). These data strongly suggest that cagE gene is, indeed, involved in inducing apoptosis of human epithelial cells in vitro.

Previous studies have shown that cytochalasin D significantly inhibited A. actitiontycetenicomitans-mediated apoptosis of KB cells, suggesting that it is dependent on the cytoskeletal function for bacterial invasion in vitro (Fives-Taylor et al.,

1999; Meyer et al., 1996). To test whether CagE-induced apoptosis was also dependent on the same process, the inventor analyzed the apoptosis of KB cells induced by A. actinomycetemcomitans in the presence of cytochalasin D at the concentrations of 0.5-2 ug/ml. The results showed that cytochalasin D, regardless the concentrations tested, had no effect on the presence and absence of apoptotic KB cells induced by either WT JP2 or cagE mutant strains, respectively (Fig. 6D). Thus, it appeared that the apoptosis induced by CagE in A. actinornycetemconaitans was independent of the cytoskeletal functions associated with bacterial invasion (Fives-Taylor et al., 1999; Meyer et al., 1996).

EXAMPLE 5: CagE gene is involved in bacterial adherence to human epithelial cells It has been shown that some Gram (-) pathogens use the type-IV secretion system for bacterial adherence to the host cells (Donnenberg, 1999). To investigate whether cagE gene is involved in bacterial adherence to the host cells, human epithelial KB cells were co-cultured with wild type (JP2) or cagE mutant, respectively.

The results showed that, compared to WT JP2 strain, cagE mutant showed a significantly reduced ability to adhere to human epithelium KB cells, at 4 hr post- treatment (Fig. 7A & 7B). As shown in Fig. 7A & 7B, there was significantly less adherence (or contact) of the cagE mutant to the KB cell surfaces, compared to the wild type (WT) JP2 strain. Therefore, these data suggest that cagE gene is involved in mediating bacterial adherence to the host cells at least in vitro.

EXAMPLE 6: N-terminal region of the CagE protein is critically involved in inducing apoptosis To localize the critical fragments involved in apoptosis, different sizes of truncated CagE proteins were expressed, purified and co-cultured with KB epithelial cells. The truncated CagE proteins were engineered as follows: N-terminal truncated CagE137-340 (SEQ ID NO: 5) (1-136 a. a. removed thus designated: N-CagE; =22 KDa); C-terminal truncated Caget 309 (SEQ ID NO: 6) (310-340 a. a. removed thus designated: Cl-CagE ; ~34 KDa); C-terminal truncated CagE,-270 (SEQ ID NO: 7) (271-340 a. a. removed thus designated: C2-CagE; =27 KDa). Expression and purification of the truncated recombinant CagE fusion proteins were performed according to the manufacturer's protocols. Briefly, SG13009 cells were grown and monitored until they reached 0.5 at OD600, then were subjected to IPTG induction for 4 hrs after which cells were pelleted and lysed in buffers. Then proteins were purified on a Ni-NTA agarose column according to the manufacturer's instructions (Qiagen, ON).

Purified proteins were subjected to a 12.5 % SDS-PAGE analysis and the 22,34, and

27-kDa CagE protein bands, respectively, were excised after brief staining with Coomassie-Blue. The truncated CagE proteins were then eluted with 50mM ammonium bicarbonate containing 0.01% SDS (pH 7.4) and further dialysed in PBS for 48 hrs.

Finally, the purified proteins were re-suspended in 50 mM PBS (pH 7.0) and stored at - 80C. Protein purity was further assessed by SDS-PAGE with Coomassie-Blue and Silver staining in which only one detectable protein band representing the respective CagE truncated protein was observed (data not shown).

The results showed that addition of the N-terminal (1-136) truncated CagE protein, not the others, did not induce morphological criteria of apoptosis in KB cells (Fig. 8, top-panel B/W photographs), suggesting that a. a. 1-136 region of the N-terminus was critically involved in inducing apoptosis of the epithelial cells. To verify the above findings at single cell level, a TUNEL assay was employed. The results showed that while significantly, higher numbers of TUNEL-positive cells were detected in Cl-CagE- treated KB cells (also in C2-CagE & whole CagE proteins), there was only background level of TUNEL-positive cells detected in NI-136trancated CagE-treated KB cells (Fig 8, middle-panel). Further quantitation of the fluorescence-labeled TUNEL assays confirmed that N-terminus was critically involved in CagE-induced apoptosis of KB epithelial cells in vitro (p<0.001 ; Fig. 8: bottom diagram).

Altogether, it is evident that purified CagE protein at pM-M concentrations induced significant apoptosis of various human cells in vitro. Further, CagE of A. actinomycetemcomitans, functions as an effector, via its N-terminal region, by inducing apoptosis of human cells.

EXAMPLE 7: Generation of single-chain Fv monoclonal antibodies Fresh HuPBL was collected by Ficoll-Hypaque using individual blood samples obtained from consenting normal healthy donors (n=5) without periodontitis and A. actinonzyctemcomitans pathogen (in dental plaque samples) at UWO dental clinic and engrafted into a group of SCID mice (n=3-5 per donor). Each HuPBL-SCID mouse was immunized i. p. with 50jig of A. actinomycetemcomitans-specific purified CagE protein in CFA/IFA (v/v=1110) adjuvant mix described previously (Teng et al, 1999; Teng et al, 2000; Nguyen et al, 2000). At day 14, human IgM & IgG in mouse sera was individually assessed by ELISA and for the one (s) with the highest IgG levels (21-5mg/ml) & titres, their spleens were pooled for mRNAs and construction of human scFv libraries (Nguyen et al, 2000). The mRNAs derived from HuPBL samples of the healthy (n=5) vs. other A. actinomycetemcomitans-infected patients (n=5) were also be used as an alternative source to ensure the best libraries constructed. Then, human Ig heavy & light chain Variable region genes (VH & VL) were amplified by RT-PCR using

human VH/VL-specific primer sets as previously described (Marks et al, 1991). The resulting VH & VL PCR amplicons were randomly linked together by oligo-peptides [-Gly4Ser-) 3], which were cloned into the pCANTAB-5E phage vector according to the manufacturer's protocol. Direct bio-panning was performed by using purified CagE protein and phage libraries (0. 5xl08pfu/well) in vitro as described previously (Nguyen et al, 2000). The strong binders (usually of higher binding affinity) were selected by using stringent washing conditions and soluble scFv's ELISA signals that are at least 102-103 times greater than that of the negative control (using total proteins of cagE' knock-out strain and a third-party antigen). The bound phages were eluted, then used to infect growing E. coli TG1 (suppressor F'strain) followed by selection on Ampicilin (+) LB agar plates. The binding phages were picked then used to infect E. coli HB2151 (suppressor F strain) to produce soluble CagE-specific scFv proteins.

FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION <BR> <BR> Arends M. , et al. , (1990) Am. J. Pathol. , 136: 593-607.<BR> <P>Asahi, M. , Azuma, T. , Ito, S. , Ito, Y. , Suto, H. , Nagai, Y. , Tsubokawa, M. , Tohyama, Tohyama,Y.,<BR> Maeda, S. , Omata, M. , Suzuki, T. , and Sasakawa, C. (2000) Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells. J Exp Med 191 (4): 593-602. <BR> <BR> <P>Censini, S. , Lange, C., Xiang, X. , Crabtree, J. E. , Ghiara, P. , Borodovsky, M. ,<BR> R. , and Covacci, A. (1996) cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc Natl Acad Sci U S A 93 (25): 14648-14653.

Chen, Y. , Smith, M. R., Thlrumalai, K, and Zychlinsky, A. (1996) A bacterial invasin induces macrophage apoptosis by binding directly to ICE. EMBO J 15 (15): 3853-3860.

Christie, P. J. , and Vogel, J. P. (2000) Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol 8 (8): 354-360.

Cotter T. , et al. , (1990) Anticancer Res. , 10: 1153-1160.<BR> <P>Covacci, A. , Telford, J. L. , Del, Giudice, G. , Parsonnet, J. , and Rappuoli, R. (1999) Helicobacterpylori virulence and genetic geography. Science 284 (5418): 1328-1333 Covacci, A. and Rappuoli, R. (2000) Tyrosine-phosphorylated bacterial proteins: Trojan horses for the host cell. J Exp Med 191 (4): 587-592.

Donnenberg, M. S. (1999) Interactions between enteropathogenic Escherichia coli and epithelial cells. Clin In-fect Dis 28 (3): 451-455.

Ebersole, J. L. , and Taubman, M. A. (1994) The protective nature of host responses in periodontal diseases. Periodontol 2000. 5: 112-141.

Fiering, S., Northrop, J. P. , Nolan, G. P. , Matilla, P. , Crabtree, G. R. , and andHerzenberg, L. A.

(1990) Single cell assay of a transcription factor reveals a threshold in transcription

activated by signals emanating from the T-cell antigen receptor. Genes Dev 4: 1823- 1834. <BR> <BR> <P>Fives-Taylor, P. M. , Meyer, D. H. , Mintz, K. P., and Brissette, C. (1999) Virulence factors of Actinobacillus actinomycetemcomitans. Periodontol 2000. 20: 136-167.

Galan, J. E. , and andCollmer, A. (1999) Type III secretion machines: bacterial devices for protein delivery into host cells. Science 284 (5418): 1322-1328.

Gerschenson L. and Rotello R. (1992) FASEB J. , 6: 2450-2455.

Hurn, B. A. L. and Chantler, Shireen M. in Methods in Enzymology Volume 70 pp 104 to 142; ed. Helen Van Vunakis and John J. Langone, 1980.

Jablonske, E. et al., (1986) Nuc. Acids Res, 14: 6115-6128.

Kato, S. , Muro, M. , Akifusa, S. , Hanada, N. , Semba, Semba,I., Fujii, T. , Kowashi, Y. ,<BR> andNishihara, T. (1995) Evidence for apoptosis of murine macrophages by Actinobacillus actinomycetemcomitans infection. Infect Immun 63 (10): 3914-3919.

Kato, S. , Nakashima, K., Inoue, M. , Tomioka, J. , Nonaka, K. , Nishihara, T. ,<BR> Kowashi, Y. (2000) Human epithelial cell death caused by Actinobacillus actinofnycetemcomitans infection. JMed Microbiol 49 (8): 739-745.

Kirby, A. C. , Meghji, S. , Nair, S. P. , White, P. , Reddi, Reddi,K., Nishihara, T. , Nakashima, K.,<BR> Willis, C. , Sim, R. and Wilson, M. (1995) The potent bone-resorbing mediator of Actinobacillus actinornycetemcomitans is homologous to the molecular chaperone GroEL. J Clin Invest 96 (3): 1185-1194.

Kohler, G. and Milstein, C. (1976), Eur. J. Immunol. 6: 511.

Kolodrubetz, D. , Phillips, L. H. , Ezzo, P. J. , and andKraig, E. (1995) Directed genomic integration in Actinobacillus actinomycetemcomitans : generation of defined leukotoxin- negative mutants. InfectImmun 63 (7): 2780-2784.

Korostoff, J. , Wang, J. F. , Kieba, Kieba,I., Miller, M. , Shenker, B. J. , and Lally, E. T. (1998) Actinobacillus actinomycetemcomitans leukotoxin induces apoptosis in HL-60 cells.

Infect Imrnun 66 (9): 4474-4483.

Landegren, et al. (1988) Science 242: 229.

Maniatis, D. et al. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor N. Y.

Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G.

(1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 1991 Dec 5; 222 (3): 581-97.

Matthews and Kricka (1988) Anal. Biochem 169: 1.

Meyer, D. H. , Lippmann J. E. , and Fives-Taylor, P. M. (1996) Invasion of epithelial cells by Actinobacillus actinomycetemcomitans : a dynamic, multistepprocess. Infect Immun 64 (8): 2988-2997.

Mills, S. D. , Boland, Boland,A., Sory, M. P. , van der Smissen, P. , Kerbourch, G. , Finlay, B. B., and Cornelis, G. R. (1997) Yersinia enterocolitica induces apoptosis in macrophages by a process requiring functional type III secretion and translocation mechanisms and involving YopP, presumably acting as an effector protein. Proc Natl Acad Sci U S A.

94 (23): 12638-41263.

Mittlin (1989) Clinical Chem. 35: 1819.

Nalbant, A., and Zadeh H. H. (2000) Evidence for apoptosis of the majority of T cells activated in vitro with Actinobacillus actinoniycetemcomitans. Oral Microbiol Imfnunol 15: 290-298. <BR> <BR> <P>Nguyen, H. , Teng, Y-T. A. (2000) Efficient generation of respiratory syncytial virus<BR> (RSV) -neutralizing human MoAbs via human peripheral blood lymphocyte (hu-PBL)- SCID mice and scFv phage display libraries. Clin & Exp Immunol, 122: 85-93.

Offenbacher, S. (1996) Periodontal diseases: Pathogenesis. Ann Periodontol 1 : 821- 878.

Odenbreit, S. , Puls, J., Sedlmaier, B. , Gerland, E. , Fischer, W. , and Haas, R. (2000) Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion. Science 287 (5457): 1497-1500.

Ogura, K. , Maeda, S. , Nakao, M. , Watanabe, T. , Tada, M. , Kyutoku, T. , Yoshida, H.,<BR> Shiratori, Y. , and Omata, M. (2000) Virulence factors of Helicobacter pylori responsible for gastric diseases in mongolian gerbil. JExp Med. 192 (11): 1601-1610.

Paju, S. , Goulhen, Goulhen,F., Asikainen, S. , Grenier, D. , Mayrand, D. , and Uitto, V. (2000) Localization of heat shock proteins in clinical Actinobacillus actinomycetemcomitans strains and their effects on epithelial cell proliferation. FEMS Microbiol Lett 182 (2): 231-235. <BR> <BR> <P>Sambrook, J. , Frittsch, E. F. , and Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.

Sanderson, S., Campbell, D. J. , and Shastri, N. (1995) Identification of a CD4+ T cell stimulating antigen of pathogenic bacteria by expression cloning. J Exp Med 182: 1751-1757. <BR> <BR> <P>Stein, M., Rappuoli, R. , and andCovacci, A. (2000) Tyrosine phosphorylation of the Helicobacter pylori CagA antigen after cag-driven host cell translocation. Proc Natl AcadSci USA. 97 (3): 1263-1268.

Stoufi, E. D. , Taubman, M. A. , Ebersole, J. L. , Smith, D. J. , and Stashenko, P. P. (1987) Phenotypic analyses of mononuclear cells recovered from healthy and diseased human periodontal tissues. J Clin Immunol 7 (3): 235-245.

Suzuki, J. B. , Park, S. K. , and andFalkler, W. A Jr. (1984) Immunologic profile of juvenile periodontitis. I. Lymphocyte blastogenesis and the autologous mixed lymphocyte response. J Periodontol 55 (8): 453-460.

Teng, Y. T. , Nguyen, H. , Gao, X. , Kong, Y. Y., Gorczynski, R. M. , Singh B. , Ellen, Ellen,R. P. and Penninger, J. M. (2000) Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection. J Clin Invest 106 (6): R59-67.

Teng, Y. T. , Nguyen, H. , Hassanloo, A., Ellen, R. P., Hozumi, N. and Gorczynski, R. M.

(1999) Periodontal immune responses of human lymphocytes in Actinobacillus actinomycetemcomitans-inoculated NOD/SCID mice engrafted with peripheral blood leukocytes of periodontitis patients. J Periodontal Res 34 (1) : 54-61.

Tummuru, M. K. , Sharma, S. A. , and Blaser, M. J. (1995) Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL-8 in gastric epithelial cells. Mol Microbiol 18 (5): 867-876 Yamashita, K. , Eastcott, J. W. , Taubman, M. A. , Smith, D. J. and Cox, D. S. (1991) Effect of adoptive transfer of cloned Actinobacillus actinomycetemcomitans-specific T helper cells on periodontal disease. Infect Immun 59: 1529-1534.

Zambon, J. J. , T. Umemoto, E. De Nardin, F. Nakazawa, L. A. Christersson, and R. J.

Genco. (1988) ActiTaobacillus actinomycetemcomitans in the pathogenesis of human periodontal disease. Adv. Dent. Res. 2: 269-274.

Zambon, J. Z. (1996) Periodontal disease: Microbial factors. Ann Periodontol 1: 879- 925.

All scientific publications and patent documents are incorporated herein by reference.

The present invention has been described with regard to preferred embodiments.

However, it will be obvious to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as described in the following claims.