Vaccine compositions and immunogenic compositions are described which are glucosyltransferase subunit vaccines for dental caries and which contain at least one peptide which corresponds to a sequence of glucosyltransferase containing aspartate 413, an equivalent of aspartate 413, aspartate 451, an equivalent of aspartate 451, aspartate 562, an equivalent of aspartate 562, aspartate 567, an equivalent of aspartate 567, histidine 561, an equivalent of histidine 561, tryptophan 491, an equivalent of tryptophan 491, glutamate 489, an equivalent of glutamate 489, arginine 449, an equivalent of arginine 449, or combinations thereof. These subunit vaccines elicit antibodies which protect an immunized mammal from dental caries. Methods of provoking an immune response to intact glucosyltransferase are also described.

DETAILED DESCRIPTION OF THE INVENTION

The principal etiologic agents of dental caries are Mutans streptococci. These oral pathogens infect the oral cavity during early childhood and normally remain associated with the host's dentition for life. Mutans streptococci must colonize and then accumulate on the tooth surface in sufficient numbers to achieve dissolution of the enamel. After the initial colonization by Mutans streptococci on the tooth surface, the Mutans streptococci produce glucosyltransferase (GTF), an enzyme which catalyzes the synthesis of glucans from sucrose. In addition, S. mutans express cell surface proteins which serve as glucan binding sites. Glucans mediate much of the subsequent accumulation of Mutans streptococci on the tooth surface. This results in an increase in the numbers of potentially cariogenic bacteria in plaque. The metabolism of various saccharides by the accumulated bacterial mass results in excretion of significant amounts of lactic acid as a metabolic product, which causes demineralization when present in sufficient amount in close proximity to the tooth surface. This eventually results in a carious lesion (a cavity).

Recently, primary and secondary structural comparisons of glucosyltransferases with the alpha amylase superfamily have provided insights into the structure-function relationships of the GTF catalytic domain. Much of the catalytic activity of alpha amylases is contained in a (.beta.,.alpha.).sub.8 barrel element (Matsuura, et al., J. Biochem. 95:697 702 (1984)). Aspartates or glutamates at the C terminus of .beta. strands (Dertzbaugh, et al., Infect. Immun. 58:70 79 (1990); Devulapalle, et al., Protein Science 6:2489 2493 (1997); Funane, et al., Biochem. 32:13696 13702 (1993)) have been specifically implicated in amylolytic activity and are invariant in these enzymes (Jenkins, et al., FEBS Lett. 362:281 285 (1995)). The overall homology between alpha amylases and GTF is low, except for a 50 60 amino acid sequence stretch near the middle of the GTF molecule (Ferretti, et al., J. Bacteriol. 169:4271 4278 (1987)) for which no catalytically involved residues have been identified. However, sequence alignment techniques (Devulapalle, et al., Protein Science 6:2489 2493 (1997); MacGregor, et al., FEBS Let. 378:263 266 (1996)) have shown significant homologies between GTFs and alpha amylase with respect to several invariant residues important to the catalytic activity of the alpha amylase family, and have suggested that the (.beta.,.alpha.).sub.8 barrel element may also be a feature of the GTF catalytic domain. Strengthening this conclusion are site-directed mutagenesis studies (Devulapalle, et al., Protein Science 6:2489 2493 (1997); Tsumori, et al., Infect. Immun. 179:3391 3396 (1997)) which showed that modification of aspartates or glutamates in GTF, which aligned with the catalytically important residues in the .beta.4, .beta.5, and .beta.7 strands of alpha amylases, drastically reduced GTF catalytic activity.

Since residues in or near the putative .beta.5 and .beta.7 strands of GTF thus appear to be functionally important, it was of interest to determine whether significant antigenic epitopes exist within these sites of GTF catalytic activity and whether antibody to these putative epitopes could inhibit enzyme activity. Under the hypothesis that sequential epitopes within these regions could be mimicked by synthetic peptides, two synthetic peptide constructs were prepared whose sequences contained the .beta.5 and .beta.7 strands, as well as adjacent residues that were implicated in catalytic activity by modeling and site-directed mutagenesis techniques (MacGregor et al., FEBS Let. 378:263 266 (1996); Tsumori et al., Infect. Immun. 179:3391 3396 (1997)). These peptide constructs were then assessed for their ability to induce serum IgG and salivary IgA antibody to peptide and to S. mutans GTF, as well as for their ability to inhibit the catalytic activity of mutans streptococcal GTF.

The compositions of the present invention, e.g., vaccine compositions and immunogenic compositions, comprise at least one peptide consisting essentially of an amino acid sequence of glucosyltransferase comprising an amino acid selected from the group consisting of aspartate 413, aspartate 451, aspartate 562, aspartate 567, histidine 561, tryptophan 491, glutamate 489, arginine 449, an equivalent of aspartate 413, an equivalent of aspartate 451, an equivalent of aspartate 562, an equivalent of aspartate 567, an equivalent of histidine 561, an equivalent of tryptophan 491, an equivalent of glutamate 489, an equivalent of arginine 449, and combinations thereof, and which is of sufficient length to raise an immune response in a mammal to whom it is administered. The desired effect of these compositions is interruption of the cariogenic process, resulting in reduction, i.e., lessening or prevention, of dental caries.

The primary sequences of several mutans streptococcal GTFs have been deduced from DNA studies (Ferretti et al., Infect. Imm. 56:1585 1588 (1988); Russell et al., J. Dental Res. 67:543 547 (1988); Uoda et al., Gene 69:1101 1109 (1988)). Although GTFs are large molecules, they function through a few discrete sites, which include the catalytic and glucan-binding sites. Primary sequences have been identified which provisionally include these sites (Mooser et al., J. Dental Res. 69:325 (1990); Russell et al., J. Dental Res. 67:543 547 (1988)).

As used herein, a vaccine composition is a composition which elicits an immune response in a mammal to which it is administered and which protects the immunized mammal against subsequent challenge by the immunizing agent or an immunologically cross-reactive agent. Protection can be complete or partial (i.e., a reduction in symptoms or infection as compared with an unvaccinated mammal). An immunologically cross-reactive agent can be, for example, the whole protein (e.g., glucosyltransferase) from which a subunit peptide used as the immunogen is derived. Alternatively, an immunologically cross-reactive agent can be a different protein which is recognized in whole or in part by the antibodies elicited by the immunizing agent.

As used herein, an immunogenic composition is intended to encompass a composition which elicits an immune response in a mammal to which it is administered and which may or may not protect the immunized mammal against subsequent challenge with the immunizing agent.

Peptides which are particularly useful in the present invention are peptides which consist essentially of an amino acid sequence of GTF comprising an amino acid selected from the group consisting of aspartate 413, aspartate 451, aspartate 562, aspartate 567, histidine 561, tryptophan 491, glutamate 489, arginine 449, an equivalent of aspartate 413, an equivalent of aspartate 451, an equivalent of aspartate 562, an equivalent of aspartate 567, an equivalent of histidine 561, an equivalent of tryptophan 491, an equivalent of glutamate 489, an equivalent of arginine 449, and combinations thereof. For example, the amino acid sequence can be the amino acid sequence of the EAW peptide (ANDHLSILEAWSDNDTPYLHD; (SEQ ID NO: 1)) or the HDS peptide (VPSYSFIRAHDSEVQDLIA; (SEQ ID NO: 2)). The invention also relates to the GLB peptide (TGARTINGQLLYFRANGVQVKG; (SEQ ID NO: 3)). Appropriate amino acid sequences will contain one or more of aspartate 413, aspartate 451, aspartate 562, aspartate 567, histidine 561, tryptophan 491, glutamate 489, arginine 449, or equivalents of these amino acids. Aspartate 413, aspartate 451, aspartate 562, and aspartate 567 refer to the aspartate residues at amino acid positions 413, 451, 562 and 567, respectively, of S. mutans GTF-B. As used herein, equivalents of these aspartate residues are intended to include catalytic aspartate residues present at equivalent sites (positions) in other mutans streptococcal GTFs (see, for example, Table 1). That is, the amino acid position numbers of the aspartate residues can be different from 413, 451, 562, and 567 in other mutans streptococcal GTFs. These equivalent aspartate residues can be identified, for example, by aligning the amino acid sequences of other streptococcal GTFs based on homology to S. mutans GTF-B using methods known in the art. In addition, the characterization of the catalytic properties of an aspartate which is equivalent to aspartate 413, 451, 562, or 567 can be determined by methods described herein or methods known in the art (see, for example, Funane et al., Biochem. 32:13696 13702 (1993)).

Similarly, histidine 561, tryptophan 491, glutamate 489 and arginine 449 refer to the histidine, tryptophan, glutamate and arginine residues, respectively, at amino acid positions 561, 491,489 and 449, respectively, of S. mutans GTF-B. As used herein, equivalents of these histidine, tryptophan, glutamate and arginine residues are intended to include histidine, tryptophan, glutamate and arginine residues, respectively, present at equivalent sites (positions) in other mutans streptococcal GTFs (see, for example, Table 1). That is, the amino acid position numbers of these residues can be different in other mutans streptococcal GTFs. These equivalent residues can be identified, for example, by aligning the amino acid sequences of other streptococcal GTFs based on homology to S. mutans GTF-B. In addition, the characterization of the properties of amino acid residues which are equivalent to, e.g., histidine 561 can be determined by methods described herein or methods known in the art (see, for example, Funane et al., Biochem. 32:13696 13702 (1993)).

Useful peptides will be of sufficient length to raise an immune response in a mammal to whom it is administered but will be less than the complete amino acid sequence of the intact GTF enzyme. Typically, the peptide will be at least 5 7 amino acids in length. Preferably the peptide will be at least 12 amino acids in length; more preferably the peptide will be at least 19, 20 or 21 amino acids in length.

The immune response which is raised can comprise a B cell response, a T cell response or both a B cell and T cell response. The B cell response is associated with the appearance of mucosal antibody, which is predominately IgA, and systemic antibody, which is predominantly IgG. The antibodies elicited by immunization will preferably recognize both the immunizing agent and an immunologically cross-reactive agent. In a preferred embodiment the antibody response will be sufficient to protect the immunized mammal against subsequent challenge or infection with the immunizing agent or an immunologically cross-reactive agent.

Although the vaccine composition of the present invention can contain one peptide consisting essentially of an amino acid sequence of glucosyltransferase comprising an amino acid selected from the group consisting of aspartate 413, aspartate 451, aspartate 562, aspartate 567, histidine 561, tryptophan 491, glutamate 489, arginine 449, an equivalent of aspartate 413, an equivalent of aspartate 451, an equivalent of aspartate 562, an equivalent of aspartate 567, an equivalent of histidine 561, an equivalent of tryptophan 491, an equivalent of glutamate 489, an equivalent of arginine 449, and combinations thereof, and which is of sufficient length to raise an immune response in a mammal to whom it is administered, preferred embodiments of the vaccine composition of the present invention contain two or more of such peptides.

Those skilled in the art will be able to determine other immunologic domains of GTF, as well as additional immunologic components of non-GTF origin which enhance adjuvanticity or produce an immunogenic response against other infectious agents, suitable for use in the vaccine composition. For example, the peptides disclosed herein can be valuably combined in a vaccine or immunogenic composition with one or more CAT, GLU, GGY, AND or SAND peptides or surface binding domain peptides such as those disclosed in U.S. Pat. No. 5,686,075 and in U.S. patent application Ser. No. 08/967,573 (Smith and Taubman), the entire teachings of which are incorporated herein by reference. In particular embodiments, the vaccine or immunogenic composition of the present invention can comprise an additional immunologic component which is an immunogenic portion of a pathogen such as, but not limited to, diphtheria, pertussis, tetanus, measles and polio virus, resulting in a multivalent vaccine producing protection against more than one infectious disease or agent. Ultimately, a multivalent vaccine can be produced which incorporates relevant protective epitopes and appropriate adjuvant sequences targeting early childhood infections.

The peptides present in the vaccine composition of the present invention may be designed in a number of ways to enhance immunogenicity. In one embodiment in which the vaccine composition contains one or more peptides, the peptide is conjugated to a known protein, (such as tetanus toxoid) or a carrier (such as a synthetic polymer carrier) to give a macromolecular structure to the vaccine which thereby enhances immunogenicity. For example, suitable peptide(s) are incorporated into a microparticle or microsphere, e.g., a PLGA (poly(lactide-co-glycolide) adjuvant) microparticle, for improved delivery and immune response. Different particles or spheres have different release profles depending on properties, such as polymer material, pore size, total particle/sphere size, and degradation kinetics. Such bioadhesive microparticles can facilitate primary and secondary mucosal antibody formation. Microparticles prepared from the biodegradable and biocompatible polymers, the poly(lactide-co-glycolides) or (PLG), have been shown to be effective adjuvants for a number of antigens. Moreover, PLG microparticles can control the rate of release of entrapped antigens and therefore, offer potential for the development of single-dose vaccines. To prepare single-dose vaccines, microparticles with different antigen release rates are combined as a single formulation to mimic the timing of the administration of booster doses of vaccine. Adjuvants can also be entrapped within the microparticles or, alternatively, adjuvants can be co-administered.

Other examples of suitable microparticles or microspheres, which can be mixed with or loaded with the proteins, peptides, or antibodies described herein, include, but are not limited to, poly(sebacic anhydride) (PSA) microspheres (Berkland et al., J. Controlled Release vol. 24 (2003)); poly(ethylene glycol)/polylactide nano-particles (Caliceti et al., J. Controlled Release vol. 24 (2003)); oligo(poly(ethylene glycol) fumarate) (OPF) (Holland et al., J. Controlled Release vol. 24 (2003))

Other suitable biocompatible, biodegradable polymers include, for example, poly(lactides), poly(glycolides), poly(lactide-co-glycolides), poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolic acid)s, polycaprolactone, polycarbonates, polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters, polycyanoacrylates, poly(p-dioxanone), poly(alkylene oxalate)s, biodegradable polyurethanes, blends and copolymers thereof.

Further, the terminal functionalities of the polymer can be modified. For example, polyesters can be blocked, unblocked or a blend of blocked and unblocked polymers. A blocked polymer is as classically defined in the art, specifically having blocked carboxyl end groups. Generally, the blocking group is derived from the initiator of the polymerization and is typically an alkyl group. An unblocked polymer is as classically defined in the art, specifically having free carboxyl end groups.

Acceptable molecular weights for polymers used in this invention can be determined by a person of ordinary skill in the art taking into consideration factors such as the desired polymer degradation rate, physical properties such as mechanical strength, and rate of dissolution of polymer in solvent. Typically, an acceptable range of molecular weights is of about 2,000 Daltons to about 2,000,000 Daltons. In a preferred embodiment, the polymer is a biodegradable polymer or copolymer. In a more preferred embodiment, the polymer is a poly(lactide-co-glycolide) (hereinafter "PLGA") with a lactide:glycolide ratio of about 1:1 and a molecular weight of about 5,000 Daltons to about 70,000 Daltons. In an even more preferred embodiment, the molecular weight of the PLGA used in the present invention has a molecular weight of about 6,000 to about 31,000 Daltons.

The microparticles or microspheres are 0.25 6.0 microns in dimension. Suitable microparticles are 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0 microns.

A sustained release composition of the invention contains from about 0.01% (w/w) to about 50% (w/w) of protein, peptide, or antibody incorporated into particles. The amount of such particles used will vary depending upon the desired effect of the protein, peptide, or antibody, the planned release levels, the times at which protein, peptide, or antibody should be released, and the time span over which the protein, peptide, or antibody will be released. A preferred range of particle loading is between about 0.1% (w/w) to about 30% (w/w) protein, peptide, or antibody to particles. A more preferred range of protein, peptide, or antibody to particle loading is between about 0.1% (w/w) to about 20% (w/w) particles. The most preferred loading of the particles is about 15% (w/w).

The sustained release composition of this invention can be formed into many shapes such as a film, a pellet, a cylinder, a disc or a microparticle A microparticle, as defined herein, comprises a polymeric component having a diameter of less than about one millimeter and having protein-, peptide-, or antibody-loaded particles dispersed therein. A microparticle can have a spherical, non-spherical or irregular shape. It is preferred that a microparticle be a microsphere. Typically, the microparticle will be of a size suitable for injection. A preferred size range for microparticles is from about 1 to about 180 microns in diameter.

A suitable polymer solution contains between about 1% (w/w) and about 30% (w/w) of a suitable biocompatible polymer, wherein the biocompatible polymer is typically dissolved in a suitable polymer solvent. Preferably, a polymer solution contains about 2% (w/v) to about 20% (w/v) polymer. A polymer solution containing 5% to about 10% (w/w) polymer is most preferred.

The method for forming a composition for modulating the release of a biologically active agent from a biodegradable polymer is further described in U.S. Pat. No. 5,656,297 to Bernstein et al. One suitable method for forming a sustained release composition from a polymer solution is the solvent evaporation method described in U.S. Pat. No. 3,737,337, issued to Schnoring et al., U.S. Pat. No. 3,523,906, issued to Vranchen et al., U.S. Pat. No. 3,691,090, issued to Kitajima et al., or U.S. Pat. No. 4,389,330, issued to Tice et al. Another method for forming sustained release microparticles from a polymer solution is described in U.S. Pat. No. 5,019,400, issued to Gombotz et al. This method of microsphere formation, as compared to other methods, such as phase separation, additionally reduces the amount of protein, peptide, or antibody required to produce a sustained release composition with a specific protein, peptide, or antibody content.

The proteins, peptides, or antibodies described herein can also be conjugated to polymers, such as N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (Nan et al., J. Controlled Release vol. 24 (2003); polyvinylpyrrolidone (PVP) (Souza et al., J. Controlled Release vol. 24 (2003)); branched poly(L-glutamic acid) attached to poly(amidoamine) (PAMAM) dendrimer or polyethyleneimine (PEI) cores (Tansey et al., J. Controlled Release vol. 24 (2003)); or bacterial polysaccharide or lipopolysaccharide (LPS) (see e.g., Frosch, M. in "Vaccine Delivery Strategies").

Additionally, other ways of enhancing immune responses to mucosally applied peptides (antigens) include use of mucosal adjuvants such as detoxified versions of tetanus toxin (e.g. tetanus toxin Fragment C), cholera toxin or E. coli heat-labile toxins (Smith et al., Infect. Immunity 69(8):4767 4773 (2002)). Other immunostimulatory adjuvants include LPS derivatives, saponins, CpG oligonucleotides, and cytokines.

In a preferred embodiment in which the vaccine composition contains at least two peptides, the peptides are synthesized and covalently attached to a peptidyl core matrix to yield a macromolecule with a high density of peptides in a single structure. Each peptide in such a structure consists essentially of an amino acid sequence of glucosyltransferase comprising an amino acid selected from the group consisting of aspartate 413, aspartate 451, aspartate 562, aspartate 567, histidine 561, tryptophan 491, glutamate 489, arginine 449, an equivalent of aspartate 413, an equivalent of aspartate 451, an equivalent of aspartate 562, an equivalent of aspartate 567, an equivalent of histidine 561, an equivalent of tryptophan 491, an equivalent of glutamate 489, an equivalent of arginine 449, and combinations thereof, and which is of sufficient length to raise an immune response in a mammal to whom it is administered. The peptidyl core matrix can consist of amino acids such as lysine, arginine and histidine. In particular, at least 2 peptides are synthesized on a core matrix of at least one lysine to yield a macromolecular vaccine composition. Particularly, at least 2 peptides are synthesized on a core matrix of 3 lysines. In a preferred embodiment, a vaccine composition is designed in which four peptides of the present invention are synthesized and covalently attached to a core matrix of 3 lysines yielding a radially branched peptide with four dendritic arms. In this embodiment, the four peptides present can be the same or different. Those skilled in the art will be able to determine other variations of synthesizing and covalently attaching vaccine compositions of the present invention to a peptidyl core matrix by employing routine experimentation.

The present invention also pertains to pharmaceutical compositions comprising at least one peptide consisting essentially of an amino acid sequence of glucosyltransferase comprising an amino acid selected from the group consisting of aspartate 413, aspartate 451, aspartate 562, aspartate 567, histidine 561, tryptophan 491, glutamate 489, arginine 449, an equivalent of aspartate 413, an equivalent of aspartate 451, an equivalent of aspartate 562, an equivalent of aspartate 567, an equivalent of histidine 561, an equivalent of tryptophan 491, an equivalent of glutamate 489, an equivalent of arginine 449, and combinations thereof, and which is of sufficient length to raise an immune response in a mammal to whom it is administered. For instance, the peptide of the present invention can be formulated with a physiologically acceptable medium to prepare a pharmaceutical composition. The particular physiological medium may include, but is not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions. The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists, and will depend on the ultimate pharmaceutical formulation desired. Methods of introduction of exogenous peptides at the site of treatment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, sublingual, intraocular and intranasal. Other suitable methods of introduction can also include rechargeable or biodegradable devices and slow release polymeric devices. The pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents.

The present invention also relates to antibodies which bind a polypeptide of the present invention. For instance, polyclonal and monoclonal antibodies, including nonhuman and human antibodies, humanized antibodies, chimeric antibodies and antigen-binding fragments thereof (Current Protocols in Immunology, John Wiley & Sons, N.Y. (1994); EP Application 173,494 (Morrison); International Patent Application WO86/01533 (Neuberger); WO 97/08320 (Morphosys) and U.S. Pat. No. 5,225,539 (Winters)) which bind to the described polypeptides are within the scope of the invention. A mammal, such as a mouse, rat, hamster or rabbit, can be immunized with an immunogenic form of the polypeptide (e.g., a peptide comprising an antigenic fragment which is capable of eliciting an antibody response). Techniques for conferring enhanced immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. The protein or polypeptide can be administered in the presence of an adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibody.

Following immunization, anti-peptide antisera can be obtained, and if desired, polyclonal antibodies can be isolated from the serum. Monoclonal antibodies can also be produced by standard techniques which are well known in the art (Kohler and Milstein, Nature 256:495 497 (1975); Kozbar et al., Immunology Today 4:72 (1983); and Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77 96 (1985)). The term "antibody" as used herein is intended to include fragments thereof, such as Fab and F(ab).sub.2. Antibodies described herein can be used to inhibit the activity of GTF, particularly in vitro and in cell extracts, using methods known in the art. Additionally, such antibodies, in conjunction with a label, such as a radioactive label, can be used to assay for the presence of the expressed protein in a cell from, e.g., a tissue sample, and can be used in an immunoabsorption process, such as an ELISA, to isolate the polypeptide or GTF protein.

The present invention further relates to a method of provoking an immune response to glucosyltransferase in a mammal by administering a peptide consisting essentially of an amino acid sequence of glucosyltransferase comprising an amino acid selected from the group consisting of aspartate 413, aspartate 451, aspartate 562, aspartate 567, histidine 561, tryptophan 491, glutamate 489, arginine 449, an equivalent of aspartate 413, an equivalent of aspartate 451, an equivalent of aspartate 562, an equivalent of aspartate 567, an equivalent of histidine 561, an equivalent of tryptophan 491, an equivalent of glutamate 489, an equivalent of arginine 449, and combinations thereof, and which is of sufficient length to raise an immune response in a mammal to whom it is administered. Preferably, the immune response results in interference with the enzymatic activity of glucosyltransferase in mammals after administration of the vaccine composition. The immune response elicited by the compositions and methods of the invention can be humoral or systemic; for example, the immune response can be a mucosal response. The immune response elicited by the method of the present invention results in reduction of the colonization or accumulation of mutans streptococcal strains in the mammal to whom the vaccine or immunogenic composition is administered.

The invention also relates to a method of immunizing a mammal against dental caries comprising administering a peptide consisting essentially of an amino acid sequence of glucosyltransferase comprising an amino acid selected from the group consisting of aspartate 413, aspartate 451, aspartate 562, aspartate 567, histidine 561, tryptophan 491, glutamate 489, arginine 449, an equivalent of aspartate 413, an equivalent of aspartate 451, an equivalent of aspartate 562, an equivalent of aspartate 567, an equivalent of histidine 561, an equivalent of tryptophan 491, an equivalent of glutamate 489, an equivalent of arginine 449, and combinations thereof, and which is of sufficient length to raise an immune response in the mammal, to the mammal.

The compositions of the present invention can be administered to any mammal in which the prevention and/or reduction of dental caries is desired. Suitable mammals include primates, humans, cats, dogs, mice, rats and other mammals in whom it is desirable to inhibit dental caries. The present invention provides a vaccine that is useful for preventing, halting or reducing the progression of dental caries in a mammal to whom the vaccine is administered.

In the method of the present invention of provoking an immune response to glucosyltransferase, mammals in which an immune response to glucosyltransferase is desired are given the vaccine or immunogenic compositions described herein. The vaccine composition can be included in a formulation which is administered to an individual being treated; such a formulation can also include a physiologically compatible carrier (e.g., a physiological buffer), stabilizers, flavorants, adjuvants and other components. The vaccine can be administered by a variety of routes (e.g., parenterally, intranasally, intraocularly, intravenously, orally) and the components of the formulation will be selected accordingly. The amount to be administered and the frequency of administration can be determined empirically and will take into consideration the age and size of the mammal being treated and the stage of the dental caries disease (e.g., prior to colonization of Mutans streptococci, soon after colonization of Mutans streptococci or in later stages of colonization).

Studies (Taubman et al., J. Dent. Res. 76:347 (1997)) indicate that multiepitopic peptide constructs induce enhanced immune responses. This strategy also could be used to increase the immune potential of the EAW/HDS/GLB peptide sequences described herein. Moreover, the combination of sequences from several strains into a synthetic or recombinant multi-epitopic construct could increase the protective potential of subunit vaccines for dental caries.

Many lines of evidence suggest that mutans glucosyltransferases require the interaction of several sequentially separated amino acid residues for their catalytic activity. Sequence alignments of GTFs with alpha-amylases have suggested that a similar (.beta.,.alpha.).sub.8 barrel structure is present in the catalytic domain of both protein families (Devulapalle, et al., Protein Science 6:2489 2493 (1997), MacGregor, et al., FEBS Let. 378:263 266 (1996)). Supporting this suggested structure is the observation that GTF activity can be significantly inhibited by site-directed mutagenesis of residues that correspond to invariant amino acids which are catalytically important in the (.beta.,.alpha.).sub.8 barrel domain of alpha amylases (Devulapalle, et al., Protein Science 6:2489 2493 (1997); Tsumori, H et al., Infect. Immun. 179:3391 3396 (1997)). The immunological findings of the present study also support the catalytic importance of residues in equivalent GTF regions. In these studies, HDS and EAW (Table 1), two peptide constructs whose sequences are adjacent to the .beta.5 and .beta.7 strand elements of mutans streptococcal GTFs, induced high levels of serum IgG and salivary IgA antibody, not only to themselves (Tables 2 and 3), but also to S. mutans GTF (FIG. 1). Furthermore, these peptides also had the ability to induce antibody which could inhibit the water-soluble glucan synthetic activity of S. mutans GTF (FIG. 2).

Alpha amylases contain three catalytic sites which are located in or adjacent to the .beta.4, .beta.5 and .beta.7 strands. Several catalytically involved amino acid residues have been implicated within analogous regions of GTF. One of these, an aspartate (Asp 451 in S. mutans GTF-B) corresponding to an invariant catalytic aspartate in the alpha amylase family (Asp 206 of taka-amylase A), has been shown by Mooser and coworkers (Mooser, et al., J. Biol. Chem. 266:8916 8922 (1991)) to be involved in glucosyl-intermediate formation by GTF. It has been reported that the synthetic peptide construct, CAT, whose sequence contains the .beta.4 strand and includes residues corresponding to the invariant Arg-449 and the above-mentioned Asp-451, can induce antibody that binds to intact GTF, significantly inhibits GTF activity, and can induce protective immunity for experimental dental caries (Smith, et al., Infect. Immunity 62:5470 5476 (1994), Taubman, et al., Infect. Immun. 63:3088 3093 (1995)). Recently, Devulapulle and Mooser (Devulapalle, et al., Protein Science 6:2489 2493 (1997)) mutated the comparable aspartate in Streptococcus downei GTF which resulted in an almost complete loss of catalytic activity.

Within the .beta.5-associated strand of alpha amylases is a glutamate residue (position 230 in taka-amylase A) which is considered to serve catalytically as a proton donor (Matsuura, et al., J. Biochem. 95:697 702 (1984)). Site-directed mutagenesis of the analogous residue in S. downei GTF (Glu-489 in S. mutans GTF-B) to glutamine resulted in a catalytically inactive enzyme (Devulapalle, et al., Protein Science 6:2489 2493 (1997)). Mutagenesis of Trp-491 in S. mutans GTF-B, highly conserved in all mutans streptococcal GTFs (Table 1), also eliminated detectable enzyme activity (Tsumori, et al., Infect. Immun. 179:3391 3396 (1997)). The EAW peptide sequence overlapped both of these important residues as well as the complete .beta.5 strand sequence. Antibody induced by the EAW peptide construct could bind to and inhibit S. mutans GTF.

The HDS peptide construct contains several residues which have been implicated in GTF function. His-561 and Asp-562 in S. mutans GTF-B are invariant in mutans streptococcal GTFs. The analogous histidine in alpha amylases helps to stabilize transition states (Sogaard, et al., J. Biol. Chem. 268:22480 22484 (1993)), while the aspartate stabilizes the reaction intermediate carbonium cation (Matsuura, et al., J. Biochem. 95:697 702 (1984)). Site directed mutagenesis of the equivalent histidine and aspartic acid residues in mutans streptococcal GTFs catalytically inactivated the enzyme (Devulapalle, et al., Protein Science 6:2489 2493 (1997); Tsumori, et al., Infect. Immun. 179:3391 3396 (1997)). Also contained within the HDS peptide sequence is an aspartate, equivalent to Asp-567 in GTF-B, which has been shown to influence the solubility of the glucan synthesized by GTF (Shimamura, et al., J. Bact. 176:4845 4850 (1994)). Aspartic acid is invariant at this position in all mutans streptococcal GTFs, although it is not conserved in alpha amylases, presumably because its function is irrelevant to amylolytic activity. Thus, antibody directed to the HDS peptide construct could be expected to influence several aspects of GTF activity. In the present study, most rats responded to HDS-peptide construct immunization with levels of antibody to GTF that were within the range of sera from rats injected with intact S. mutans GTF. Many of these sera also inhibited the water-soluble glucan synthetic activity of S. mutans GTF which is consistent with the presence of putative functional residues within this sequence.

Peptide-injected rat sera did not detectably inhibit water-insoluble glucan synthesis under the conditions of the assay. This lack of water-insoluble glucan inhibition may be related to the expected lower affinity and avidity of the anti-peptide antibody or be a consequence of assay conditions, such as the mixture of S. mutans GTF isotypes used for synthesis or the lack of primer dextran. Interestingly, antisera to intact S. mutans GTF also were less effective as inhibitors of water-insoluble, compared with water-soluble, glucan synthesis.

The MAC peptide construct was selected for control purposes, because its sequence (amino acids 342 356) lay outside the GTF (.beta.,.alpha.).sub.8 barrel domain predicted by MacGregor and coworkers (MacGregor et al., FEBS Let. 378:263 266 (1996)) or within a non-catalytically implicated approximately 200 residue loop within the (.beta.,.alpha.).sub.8 barrel domain of GTF predicted by Devulapalle and Mooser (Devulapalle et al., Protein Science 6:2489 2493 (1997)). Also this sequence bore no homology with sequences associated with catalytic function by biochemical (Funane et al., Biochem. 32:13696 13702 (1993); Mooser et al., J. Biol. Chem. 266:8916 8922 (1991)) or molecular genetic techniques (Chia et al., Immun. 61:4689 4695 (1993); Devulapalle et al., Protein Science 6:2489 2493 (1997); Tsumori et al., Infect. Immun. 179:3391 3396 (1997)). Neither serum IgG nor salivary IgA antibody to the HDS peptide construct showed any reactivity with the MAC peptide (Table 2). The MAC peptide construct was less immunogenic and induced less GTF-inhibitory antibody than did the HDS or EAW constructs, further supporting the catalytic significance of the residues within the latter two peptide sequences. Interestingly, a peptide sequence corresponding to MAC was immunogenic when fed (Dertzbaugh et al., Infect. Immun. 58:70 79 (1990)) or injected (Dertzbaugh and Macrina, Immun. 58:1509 1513 (1990)) as a protein chimera, fused to the sequence of the B subunit of cholera toxin (CTB). This difference in reactivity between the chimeric protein (Dertzbaugh and Macma, Immun. 58:1509 1513 (1990)) and that of the MAC peptide construct could be because the former had highest homology with S. mutans GTF-B and GTF-C, while the MAC peptide in the present study was identical to the respective sequences in S. sobrinus and S. downei GTF-I (Table 1). In addition, the fusion with CTB undoubtedly influenced the immunogenicity of the protein chimera.

Thus, these data indicate that sequences containing functionally important residues associated with the .beta.5 and .beta.7 barrel elements are immunogenic and can induce systemic and mucosal antibody responses that can lead to loss of enzyme function. It has been shown that antibody levels induced by other catalytically associated peptides can be increased by combination with functionally associated GTF peptides that also contain a strong T cell epitope (Taubman et al., abstr. 2666, p. 347, In J. Dent. Res. 76 (1997)). Combination of HDS and or EAW with such peptides may also enhance immune responses to these important epitopes. Since both EAW and HDS peptide constructs also gave rise to significant levels of salivary IgA antibody in many animals, di- or multi-epitopic constructs could be expected to also increase mucosal immunity, thus potentiating their application as subunit vaccines for dental caries.

 

Claim 1 of 4 Claims

1. An immunogenic composition comprising a biocompatible microparticle and at least one peptide which is an amino acid sequence subunit of glucosyltransferase of 15 22 amino acids in length comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 15; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; and SEQ ID NO: 19.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.