WO2006099101A1 - Immunogenic compositions for mucosal delivery - Google Patents

Abstract

The present invention provides for compositions and methods for interfering with colonization of mucosal surfaces by mutans streptococci. In particular, the present invention provides for the inhibition of attachment and enzymatic functions of mutans streptococci. The present invention also provides for recombinant immunogenic compositions, mucosal delivery vaccines for dental caries, and diagnostics for the identification of mutans streptococci.

Description

IMMUNOGENIC COMPOSITIONS FOR MUCOSAL DELIVERY

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT This invention was produced in part using funds obtained through grants from the

National Institutes of Health. Consequently, the federal government may have certain rights in this invention.

CROSS REFERENCE TO RELATED APPLICATION The present application is related to and claims the benefit, under 35 U.S.C. § 119(e), of U.S. provisional patent application Ser. No. 60/660,472, entitled "Immunogenic Compositions for Mucosal Delivery," filed 11 March 2005, which is incorporated by reference entirely herein.

FIELD OF THE INVENTION

The present invention relates generally to the fields of microbiology and immunology. The present invention also relates to compositions and methods for interfering with colonization of mucosal surfaces. In particular, the present invention provides for the inhibition of attachment and enzymatic functions of mutans streptococci. The present invention also provides for recombinant immunogenic compositions, mucosal delivery vaccines for dental caries, and diagnostics for the identification of mutans streptococci.

BACKGROUND OF THE INVENTION Public awareness of emerging infectious diseases, the threat of biological warfare, and the continuation of infectious disease as the leading cause of morbidity and mortality have fostered an urgent need for better means of prevention and treatment. Evidence that most infectious agents cause disease by colonizing or penetrating mucosal surfaces has prompted studies of mucosal vaccination strategies aimed at protecting the mucosae, as well as surfaces bathed by mucosal secretions, e.g., teeth. Systemic and mucosal responses are elicited and regulated somewhat independently.

For example, stimulation of the common mucosal immune system results in the appearance of secretory IgA in various mucosal secretions where it provides a first line of defense for the mucosal surfaces. Although much of our understanding of the common mucosal immune system has resulted in experimental work in animals, the human common mucosal immune system displays distinctive characteristics that may limit the applicability of information obtained from non-human models.

Dental caries is an infectious disease considered the most prevalent and costly disease in developing as well as industrialized countries. The bacterial group comprising mutans streptococci (including Streptococcus mutans and Streptococcus sobrinus) is perhaps the most crucial pathogenic factor in dental caries in human beings. Because of the potential importance of secretory IgA antibodies in protection against oral disease, including mutans streptococci-induced dental caries, there remains a need for immunogenic constructs and vaccine delivery systems that may provide further insight into the function of the common mucosal immune system in this disease model. Indeed, there remains a need for new vaccine strategies aimed at the common mucosal immune system that would be beneficial in developing approaches to protect against other common as well as emerging infectious diseases.

SUMMARY OF THE INVENTION

An object of the present invention provides for a composition and method for eliciting a common mucosal immune system response to a composition comprising a recombinant immunogen delivered via mucosal vaccination. Another object of the invention provides for the induction of secretory immune responses in adults, children, or the elderly after mucosal immunization with a composition comprising a recombinant chimeric protein, SBR-GLU. This chimeric protein represents functional regions of two virulence factors of mutans streptococci: AgSJU and glucosyltransferase. A further object of the invention provides for a method of interfering with the enzymatic activity of streptococcal glucosyltransferase.

The invention also provides for a vaccine against dental caries. An aspect of this invention provides for a vaccine against mutans streptococci, including Streptococcus mutans and Streptococcus sobrinus. Another aspect of this invention provides for the vaccine to be delivered to the mucosae of adults, children, elderly adults, or other populations in need thereof.

Another object of the invention provides for a method of characterizing how mutans streptococci become established on newly erupting permanent molar teeth. A further aspect of the invention provides for methods for determining the ability of an immune response against SBR-GLU antigens to interfere with colonization of newly erupting permanent molar teeth.

A further object of the invention relates to composition and methods that shift the

5 population that shapes the normal flora in the mouth of humans.

This invention also provides for a method of increasing ThI responses and cell- mediated immunity, wherein the method comprises administering mucosally to a subject a recombinant immunogen comprising a chimeric SBR-GLU protein. In an aspect of the invention, mucosal delivery of a composition comprising the chimeric SBR-GLU protein

0 induces a TLR 4 signal response, and results in an up-regulation of B7-1, B7-2, and CD40, as well as the induction of TNF-α, IL-10, and IL-12p40 production.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a plasmid map showing the construction of the SBR-GLU chimeric [ 5 protein expression vector.

FIGS. 2 A and 2B depict saliva IgA anti-SBR or anti-GLU responses, respectively, in mice following IN immunization with SBR, GLU, SBR+GLU, and SBR-GLU. Values are expressed as the geometric mean of the percent anti-SBR or anti-GLU IgA per total IgA (+SD) for six mice. The asterisk indicates a significant difference from the control group .0 at pO.001.

FIG. 3 depicts the percentage of S. mutans per total oral streptococci in the oral cavity of immunized or control mice challenged with 2 x 10⁹ CFU on 5 consecutive days. The values are expressed as the geometric mean + S.E. for six mice. The asterisk indicates a significant difference from the control at p<0.05.

25 FIG. 4 depicts a mucosal and oral vaccination schedule in an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION It should be understood that this invention is not limited to the particular 0 methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. As used herein and in the claims, the singular forms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise. Thus, for example, the reference to an antigen is a reference to one or more such antigens, including equivalents thereof known to those skilled in the art. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean ±1%.

All patents and other publications identified are incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the invention, the preferred methods, devices, and materials in this regard are described here.

Dental caries remains a global problem even though it appears to be on a decline in some developed countries. See, e.g., Moller, "Caries status in Europe and predictions of future trends," 24 Caries Res. 381-396 (1990). This infectious disease is considered the most prevalent and costly disease in developing as well as industrialized countries. Centers for Disease Control and Prevention, "Guidelines for school health programs to promote lifelong healthy eating," vol. 45 (RR-9) (1996). Many age groups would benefit from caries prevention, including adults, children, the elderly, or other populations in need thereof. Among children, dental caries (tooth decay) is the single most common chronic childhood disease — 5 times more common than asthma and 7 times more common than hay fever. Over fifty percent of 5- to 9-year-old children have at least one cavity or filling, and that proportion increases to seventy-eight percent among 17-year-olds. U.S. Dept. of Health and Human Servs., "Oral Health in America: A Report of the Surgeon General — Executive Summary," Nat'l Institute of Dental and Craniofacial Research, Nat'l Institutes of Health (2000). Hence, children represent a population that would greatly benefit from the invention provided herein.

The elderly present a further population that would benefit from the approach of the present invention. Tooth decay has become less common in young and middle-aged adults since the 1970s, but older adults still have as much tooth decay as they did in the 1970s. Indeed, although tooth decay dropped twenty-seven percent from the early 1970s to the early 1990s, this decrease was among adults aged 18 to 45. Decay rates remained the same among older adults ~ those aged 46 to 65. See J. Am. Dental Assoc. (July 2002). This has implications on the health of this population. For example, it has been shown that there is an association between periodontal disease and chronic obstructive pulmonary disease, the fourth leading cause of death in the United States. This link is explained, in part, by an overreaction of the inflammatory process that leads to destruction of connective tissue. This tissue destruction is present in both periodontal disease and emphysema. The disease state of the mouth may also contribute to the colonization of dental plaque by respiratory pathogens followed by aspiration into the lung. Mojon, "Oral Health and Respiratory Infection," 68(6) JCDA 340-5 (2002). This problem may be exacerbated after stroke or in cases of dementia, or other cases where oral hygiene is further compromised. Investigators have found that patients are at higher risk for pneumonia if they have dental plaque or certain types of mouth bacteria. Bryant, Reuters Health.

Additionally, other medical conditions may lead to increased risk for oral disease. See, e.g., Klassen & Krasko, "The Dental Health Status of Dialysis Patients is poor and reqμires greater attention," JCDA (2002). These populations would benefit from preventive treatment such as a vaccine against dental caries. Because dental caries occurs on tooth surfaces bathed by saliva, secretory IgA

(S-IgA) is a first line of defense against the mutans streptococci (i.e., S. mutans and & sobrinus, bacteria associated with caries initiation), as well as most pathogens since they cause disease by invasion or colonization of mucosal surfaces. Therefore, studies aimed at the development of a caries vaccine have focused on the induction of S-IgA antibodies by stimulation of the common mucosal immune system (CMIS). Michalek & Childers,

"Development and outlook for a caries vaccine," 1 Crit. Rev. Oral Biol. Med. 37-54 (1990).

Most of the understanding of the CMIS has been obtained from the study of inductive sites, such as the gut-associated lymphoid tissue (GALT) and bronchial-associated lymphoid tissue (BALT), and effector sites, such as salivary, lacrimal and mammary glands and intestinal lamina propria. See, e.g., Brandtzaeg, "Overview of the mucosal immune system," 146 Curr. Topics Microbiol. Immunol. 13-25 (1989). For GALT, lumenal antigens are taken up by specialized epithelial M cells, and are delivered to the underlying immunocompetent cells for the generation of antigen-sensitized, IgA-committed B cells. These cells leave the GALT, migrate to the mesenteric lymph nodes, enter the blood circulation, and then populate various effector sites (e.g., salivary glands), where terminal differentiation into IgA-secreting plasma cells occurs. McGhee et al., "Regulation of IgA synthesis and immune responses in external secretions," 9 J. Clin. Immunol., 175-199 (1989). Although most evidence has been generated in experimental animals, the CMIS also operates in humans. See, e.g., Czerkinsky et al., "IgA antibody-producing cells in peripheral blood after antigen ingestion: Evidence for a common mucosal immune system in humans," 84 Proc. Natl. Acad. Sci. USA 2449-2453 (1987). The CMIS, however, does not result in the uniform distribution of mucosal IgA precursor B cells, because the levels of specific S-IgA antibodies to various antigens relative to the total S-IgA concentration, and the proportions of specific S-IgAl and S-IgA2 antibodies are not the same in all secretions. Thus, gut-associated lymphoid tissue and other inductive sites (e.g., Waldeyer's ring) may preferentially supply IgA-committed, antigen-sensitized cells to restricted mucosal regions (e.g., salivary glands). See, e.g., Moldoveanu et al. "Compartmentalization within the common mucosal immune system," in Advances in Mucosal Immunology, 97-101 (Mestecky et al., eds., Plenum Press Pub. Corp., New York, N.Y., 1995).

The mutans streptococci have been implicated as the major etiologic agent involved in the initiation of dental caries. This bacterium has many cell wall-associated proteins (including, e.g., GTF enzymes and the surface protein Agl/II) which mediate attachment, polysaccharide production, metabolism and other functions involved with the pathogenicity of this organism. S-IgA antibodies are known to inhibit the adherence and accumulation of mutans streptococci on tooth surfaces and to confer protection against caries in animal models. See, e.g., Michalek & Childers, 1990.

The demonstration that mutans streptococci is the principal etiologic agent of dental caries in humans and experimental animals led to numerous investigations to determine the nature of the antigen(s) and the mode of immunization most effective in inducing immune responses which protect the host from dental caries (reviewed in Michalek & Childers, 1990). Studies in the 1970's demonstrated that local injection of killed S. mutans whole cells in complete Freund's adjuvant into the salivary gland region of rats resulted in a local salivary antibody response, which correlated with protection against dental caries. It would be unlikely that this method of immunization would be used in humans, however, because of local inflammation. The evidence that oral administration (and, more recently, nasal administration) of antigen results in the stimulation of lymphoid cells in GALT and subsequent S-IgA antibody responses in external secretions via the CMIS suggested a practical and safe approach for inducing salivary S-IgA antibodies protective against dental caries.

Therefore, studies in experimental rodent models were initiated using purified antigens of S. mutans to determine whether oral/nasal administration induced salivary S-IgA responses, which protected against dental caries. Fontana et al., "Intranasal immunization against dental caries with a Streptococcus mutans-ettήched fimbrial preparation," 6 CIm. Diagn. Lab. Immunol. 405-409 (1999); Hajishengallis & Michalek "Current status of a mucosal vaccine against dental caries," 14 Oral Microbiol. Immunol. 1-20 (1999); Hajishengallis et al., "Comparison of an adherence domain and a structural region of

Streptococcus mutans antigen I/II in protective immunity against dental caries in rats after intranasal immunization," 66 Infect. Immun. 1740-1743 (1998).

More recently, recombinant antigens from the mutans streptococci have been derived and used in animal models to determine their effectiveness in inducing protective responses against S. mutans colonization and dental caries development. Huang et al., "Induction of protective immunity against Streptococcus mutans colonization after mucosal immunization with attenuated Salmonella enterica serovar Typhimurium expressing an S. mutans adhesin under the control of in vivo-inducible nirB promoter," 69 Infect. Immun. 2154-2161 (2001); Jespersgaard et al., "Protective immunity against Streptococcus mutans infection hi mice after intranasal immunization with the glucan-binding region of S. mutans glucosyltransferase," 67 Infect. Immun. 6543-6549 (1999); Jespersgaard et al., "Effect of attenuated Salmonella enterica serovar Typhimurium expressing a Streptococcus mutans antigen on secondary responses to the cloned protein," 69 Infect. Immun. 6604-6611 (2001); Zhang et al., "Enhanced immunogenicity of a genetic chimeric protein consisting of two virulence antigens of Streptococcus mutans and protection against infection," 70 Infect. Immun. 6779-6787 (2002).

In this regard, a genetic chimeric protein has been derived that includes the salivary binding region of Agl/II (SBR) and the glucan binding region of GTF (GLU) and shown to induce greater responses and protection against infection than SBR or GLU alone. Zhang et al., 2002. Thus, there is an advantage to using a chimeric protein composed of regions from two virulence factors of S. mutans which have been shown (in mice) to induce antibodies that block the functional activity of the native protein. Jespersgaard et al. "Functional and immunogenic characterization of two cloned regions of Streptococcus mutans glucosyltransferase-I," 67 Infect. Immun. 810-816 (1999); and Yu et al., "Effects of antibodies against cell surface protein antigen PAc-glucosyltransferase fusion proteins on glucan synthesis and cell adhesion of Streptococcus mutans," 65 Infect. Immun. 2292-2298 (1997). This approach will allow the induction of antibodies that can interfere with two stages of the disease process.

A number of studies have investigated naturally occurring anti-5. mutans antibodies in humans to determine the nature of the S. mutans antigens important in caries immunity. Studies have provided evidence for the role of salivary S-IgA antibodies in regulating colonization of mutans streptococci in humans. Gregory et al., "Prevention of Streptococcus mutans colonization by salivary IgA," 5 J. Clin. Immunol. 55-62 (1985). Based on these findings and experimental animal studies showing the effectiveness of oral vaccines, studies were performed to investigate the CMIS in humans by using oral vaccines containing mutans streptococcal antigens. Early studies showed that oral administration of killed whole cells of S. sobrinus to four human volunteers resulted in the parallel induction of specific S-IgA antibodies in saliva and tears, but not in serum. Mestecky et al., "Selective induction of an immune response in human external secretions," 61 J. Clin. Invest. 731-737 (1978).

Regarding studies in humans, one study reported that fourteen volunteers ingesting GTF from S. sobrinus with aluminum phosphate had significantly higher salivary S-IgA anti- GTF activity and lower mean log ratios of S. mutans to total streptococci than seen in control subjects. Smith & Taubman, "Oral immunization of humans with Streptococcus sobrinus glucosyltransferase," 55 Infect. Immun. 2562-2569 (1987). In a separate set of studies, this group reported that individuals immunized by local application of GTF to minor secretory glands had antibody levels which were not significantly different from that seen in control subjects; however, topically immunized subjects showed significantly slowed recolonization by the indigenous S. mutans. Smith & Taubman, "Effect of local deposition of antigen on salivary immune responses and reaccumulation of mutans streptococci," 10 J. Clin. Immunol. 273-281 (1990). Other studies in humans have investigated the ability of oral vaccines consisting of liposomes containing purified S. tnutans antigens to induce salivary S-IgA responses. These results and those of others indicate that ingested S. mutans antigen stimulates IgA-precursor B cells which migrate from GALT via the peripheral blood to mucosal sites and secrete specific polymeric S-IgA antibodies in saliva, providing further evidence for the existence of the CMIS in humans, and suggest that oral immunization with S. mutans antigen may be an effective method for preventing S. mutans-induced dental caries in humans. Childers et al., "Mucosal and systemic responses to an oral Hposome-Streptococcus mutans carbohydrate vaccine in humans," 3 Reg. Immunol. 289-296 (1991); Childers et al., "Oral immunization of humans with dehydrated liposomes containing Streptococcus mutans glucosyltransferase induces salivary immunoglobulin A2 antibody responses," 9 Oral Microbiol. Immunol. 146- 153 (1994); and Czerkinsky (1987). More recent studies have focused on nasal immunization. Mucosal S-IgA responses in humans following oral immunization with antigens from S. mutans have been observed, but the magnitude of immune responses has been low and their persistence limited. Childers & Michalek, "Characterization of human immune responses to oral Ivposomal-Streptococcus mutans carbohydrate vaccine," in 1 Frontiers of Mucosal Immunology, 605-606 (Tsuchiya et al., eds., Excerpta Medica, Amsterdam 1991); Childers et al., 1994; Smith & Taubman, 1997.

Recently, there has been much interest in determining the importance of Waldeyer's ring as an induction site for mucosal responses, especially in the upper respiratory tract and oral cavity. Waldeyer's ring (consisting of palatine, lingual and nasopharyngeal (adenoids) tonsils) located at the beginning of the digestive and respiratory tracts, is continually exposed to inhaled and ingested antigens and appears to contribute IgA precursor cells particularly to the upper respiratory and digestive tracts. Kuper et al., "The role of nasopharyngeal lymphoid tissue," 13 Immunol. Today 219-224 (1992). The unique architecture of the tonsils resembles that of lymph nodes and GALT in having antigen-presenting cells, T, B, and IgG- and IgA- containing plasma cells present in characteristic regions and deep branched crypts which increase the surface area for trapping environmental materials.

Supporting evidence for the involvement of tonsils in the induction of mucosal immune responses includes: (a) intracellular J chain, a marker of pig A synthesis, in cultured tonsillar cells (Korsrud & Brandtzaeg, "Immune systems of human nasopharyngeal and palatine tonsils: Histomorphometry of lymphoid components and quantification of immunoglobulin-producing cells in health and disease," 39 Clin. Exp. Immunol. 271-280 (1980); Korsrud & Brandtzaeg "Immunohistochemical evaluation of J-chain expression by intra- and extra-follicular immunoglobulin-producing human tonsillar cells," 13 Scand. J. Immunol. 271-280 (1981)) and their secretion of plgA (Kutteh et al., "Tissue origins of human polymeric and monomelic IgA," 128 J. Immunol. 990-995 (1982)); (b) the predominance of IgAl, typical of the upper respiratory and digestive tracts (Crago et al., "Distribution of IgAl-, IgA2 and J chain-containing cells in human tissues," 132 J. Immunol. 16-18 (1984); Kett et al., "Different subclass distribution of IgA-producing cells in human lymphoid organs and various secretory tissues," 136 J. Immunol. 3631-3635 (1986)); and (c) reduced nasopharyngeal antibody responses to perorally administered live poliovirus in tonsillectomized children and their decreased nasopharyngeal resistance due to diminished S- IgA levels (Ogra, "Effect of tonsillectomy and adenoidectomy on nasopharyngeal antibody response to poliovirus," 284 N. Engl. J. Med. 59-64 (1971)).

Intranasal (IN) immunization studies in various annual models with liposomal associated antigens have resulted in encouraging results in that immunization increases antigen-specific antibody responses in pulmonary and oral secretions. Abraham, "Intranasal immunization with bacterial polysaccharide containing liposomes enhances antigen-specific pulmonary secretory antibody response," 10 Vaccine 461-468 (1992); Abraham & Shah, "Intranasal immunization with liposomes containing IL-2 enhances bacterial polysaccharide antigen-specific pulmonary secretory antibody response," 149 J. Immunol. 3719-3726 (1992); Aramaki et al., "Activation of systemic and mucosal immune response following nasal administration of liposomes," 12 Vaccine 1241-1245 (1994); Brownlie et al., "Stimulation of secretory antibodies against Bordetella pertussis antigens in the lungs of mice after oral or intranasal administration of liposome-incorporated cell-surface antigens," 14 Microb. Pathog. 149-160 (1993); de Haan et al., "Mucosal immunoadjuvant activity of liposomes: Induction of systemic IgG and secretory IgA in mice by intranasal immunization with an influenza subunit vaccine and coadministered liposomes," 13 Vaccine 155-162 (1995); de Haan et al., "Induction of a secretory IgA response in the murine female urogenital tract by immunization of the lungs with liposome-supplemented viral subunit antigen," 13 Vaccine 613-616 (1995); de Haan et al., "Liposomes as an immunoadjuvant system for stimulation of mucosal and systemic antibody responses against inactivated measles virus administered intranasally to mice," 13 Vaccine 1320-1324. (1995); Guink et al., "Intranasal immunization with proteoliposomes protects against influenza," 7 Vaccine 147-151 (1989). Encouraging results have also been observed with soluble antigens. Hameleers et al., "Mucosal and systemic antibody formation in the rat after intranasal administration of three different antigens," 69 Immunol. Cell Biol. 119-125 (1991); Hirabayashi et al., "Comparison of intranasal inoculation of influenza HA vaccine combined with cholera toxin B subunit with oral or parenteral vaccination," 8 Vaccine 243-248 (1990); Katz et al., "Protective salivary immunoglobulin A responses against Streptococcus mutans infection after intranasal immunization with S. mutans Antigen I/II coupled to the B subunit of cholera toxin," 61 Infect. Immun. 1964-1971 (1993); Mallett et al., "Intranasal or intragastric immunization with proteosome-,Sfø/ge//α lipopolysaccharide vaccines protects against lethal pneumonia in a murine model of Shigella infection," 63 Infect. Immun. 2382-2386 (1995); Orr et al.,

"Immunogenicity and efficacy of oral or intranasal Shigella flexeri 2a and Shigella sonnei proteosome-lipopolysaccharide vaccines in animal models," 61 Infect. Immun. 2390-2395 (1993); Russell et al., "Salivary, nasal, genital, and systemic antibody responses in monkeys immunized intranasally with a bacterial protein antigen and the cholera toxin B subunit," 64 Infect. Immun. 1272-1283 (1996); Takahashi et al., "Intranasal immunization of mice with recombinant protein antigen of Streptococcus mutans and cholera toxin B subunit," 35 Arch. Oral Biol. 475-477 (1995); Wu & Russell "Induction of mucosal immunity by intranasal application of a streptococcal surface protein antigen with the cholera toxin B subunit," 61 Infect. Immun. 314-322 (1993); Zang et al., 2002. Researchers have also investigated the feasibility of inducing mucosal responses in human adults via intranasal (IN) administration of liposomal S. mutans preparation containing GTF and some Agl/II (designated C-GTF). Childers et al., "Humans immunized with Streptococcus mutans antigens by mucosal routes," 81 J. Dent. Res. 48-52 (2002); Childers et al., "Nasal immunization of humans with dehydrated liposomes containing Streptococcus mutans antigen," 12 Oral Microbiol. Immunol. 329-335 (1997); Childers et al., "A controlled clinical study of the effect of nasal immunization with a Streptococcus mutans antigen alone or incorporated into liposomes on induction of immune responses," 67 Infect. Immun. 618-623 (1999).

It has been shown that IN immunization results in anti-C-GTF responses in nasal wash and saliva. Although IN immunization against respiratory pathogens (e.g., influenza and parainfluenza viruses) in humans results in responsive antibodies in nasal secretions and serum (Brown &Mestecky, "Immunoglobulin A subclass distribution of naturally occurring salivary antibodies to microbial antigens," 49 Infect. Immun. 459-462 (1985); Clements & Murphy, "Development and persistence of local and systemic antibody responses in adults given live attenuated or inactivated influenza A virus vaccine," 23 J. Clin. Microbiol. 66-72 (1986)), until fairly recently the appearance of antibodies in saliva after ESf immunization of humans has not been reported. Childers et al., 1997. At birth, the human gastrointestinal tract (including the oral cavity) is sterile. The neonate is immediately exposed to commensal as well as potentially pathogenic microbes. Salivary antibodies are not present at birth but begin to develop during the first few months of life and reach adult levels by age seven. For the neonate, protection from pathogens comes from placentally transferred antibodies, antibodies in breast milk, and non-immune factors (e.g., lactoferrin, lysozyme, and peroxidases).

The oral cavity develops a unique, dynamic microbiota with a succession of colonization beginning predominantly with organisms that are able to survive and multiply in a desquamating environment, e.g., Streptococcus salivarius and Streptococcus mitis. Once teeth begin to emerge (usually at four to eight months of age), a shift occurs in which organisms that can colonize tooth surfaces are added to the ecosystem (e.g., organisms that can form and survive in dental plaque; Streptococcus sanguis and mutans streptococci). Carlsson et al., "Lactobacilli and streptococci in the mouth of children," 9 Caries Res. 333- 339(1975); Smith et al., "Salivary IgA antibody to oral streptococcal antigens in predentate infants," 5 Oral Micro. Immunol. 57-62 (1990). Although mutans streptococci can colonize the oral cavity once teeth emerge, data supports the concept of a "window of infectivity" that occurs later. In a longitudinal study of forty-six infants from birth to age fifty-six months, it was shown that thirty-eight acquired mutans streptococci at a median age of twenty-six months. Whether this lag time between emergence of teeth and colonization depends on particular teeth (e.g., primary molars) that harbor cariogenic bacteria more readily, the oral cavity is potentially exposed to mutans streptococci before the "window of infectivity" is open. These investigators hypothesize that newly emerged teeth offer a "virgin habitat", which if not colonized by cariogenic bacteria while the window is open, is less susceptible to later colonization because of competition from other organisms that become established on the teeth. Caufield et al., "Initial acquisition of mutans streptococci by infants: Evidence for a discrete of window infectivity," 72 J. Dent. Res. 37-45 (1993).

Although the concept of a window of infection has been questioned by other investigators who detected S. mutans much earlier, the erupting tooth cannot be colonized until it emerges into the oral cavity. Milgrom et al., "Dental caries and its relationship to bacterial infection, hypoplasia, diet, and oral hygiene in 6- to 36-month-old children," 28 Community Dent. Oral Epidemiol. 295-306 (2000); Mohan et al., "The relationship between bottle usage/content, age and number of teeth with mutans streptococci colonization in 6-24- month old children," 26 Community Dent. Oral Epidemiol. 12-20 (1998).

Therefore, the period between initial tooth emergence and colonization is critical for the host to exert any influence in regulating what organisms become established. In regard to specific antibodies to oral bacteria, researchers have shown the appearance of salivary IgA antibody to early colonizers of the oral cavity (i.e., S. mitis) by twelve weeks of age. Smith et al. (1990). Hence, children should be able to respond to a mucosal vaccine with the induction of salivary antibodies.

In addition to immunization in infants, which may be a target population for vaccination against childhood diseases including dental caries, it is also relevant to consider immunization studies in preadolescent children, i.e., before the eruption of permanent second molar teeth. This concept takes into consideration the possibility of a "reopening of the window of infectivity" at times which correlate with susceptible teeth (i.e., permanent 2nd molars). This approach may provide data collection on the mucosal immune system and on safety in children, and may support the potential efficacy of this approach in preventing colonization of newly erupted teeth of infants. The studies completed in adults using native S. mutans antigens provide adequate safety data for beginning studies in this population.

Furthermore, a study in preschool aged children may provide similar data for permanent first molar teeth as data supporting safety and immunogenicity is accumulated in a younger population. This approach is useful in facilitating the ability to conduct studies in sequentially younger populations of human volunteers and could also support studies in infants. An additional population for which vaccination against caries may be appropriate are the elderly. As adults live longer and as their immune systems continue to change as they age, geriatric populations could benefit from enhanced mucosal resistance to mutans streptococci. Hence, a aspect of the present invention provides for an immunogenic composition for delivery to the mucosae of adults, children, and elderly adults. Mucosal immunology impacts on many areas of human health, and an improved understanding of the human CMIS and how it can be exploited specifically to develop protection against pathogens at mucosal surfaces contributes greatly to the achievement of goals outlined for oral health in the NIH report, "Healthy People 2010" (ht1p://wλvw.healthypeople.gov/document/html/objectives/21-01.1itm). In the context of oral health, dental caries is included as an important infectious disease to which an efficacious mucosal immune response may contribute to protection. Previous studies in adults have provided important information to support the safe and effective use of a mucosal vaccine for the induction of salivary antibody responses, which could afford protection against dental caries, however; studies in children must be initiated to understand the CMIS in this age group. The invention presented herein provides for the effectiveness of mucosal immunization with a recombinant mutans streptococci (MS) vaccine, not only in adults and elderly adults but in preadolescent and preschool aged children, in inducing MS immune responses in the oral cavity that are potentially protective against MS infection. This invention also provides for a vaccine against dental caries. Additionally, this invention provides for important information that should apply for the use of other mucosal vaccines in adults, children, and geriatric adults.

The immunogenicity and safety of native S. mutans antigens alone or in liposomes when given by a mucosal route for the induction of salivary responses has been evaluated. These studies were carried out with FDA exemption (BB-IND 2439). Four studies involved oral immunization and three studies involved IN immunization. All but one study involved protein antigens of S. mutans (i.e., one oral study immunized with cell wall carbohydrate antigen of S. mutans). Childers et al. (1991). These studies have shown that the culture supernatant of S. mutans strain GS-5 grown hi chemically defined media is enriched for GTF, and since 60% saturated ammonium sulfate precipitation resulted in a predominant protein band (~165 kDa) on SDS-PAGE which was enzymatically active hi the presence of sucrose and immunogenic when given to animals and humans, this antigen preparation has been a useful tool for mucosal immunization studies. Additionally, GS-5 is known to produce a truncated Agl/II (~155 kDa) that is released into culture medium. The results of the biochemical and immunological analysis indicate that a truncated form of Agl/II and GTF are present in the GS-5 preparation (C-GTF). Murakami et al., "Identification of a frameshift mutation resulting in premature termination and loss of cell wall anchoring of the PAc antigen of Streptococcus mutans GS-5," 65 Infect. Immun. 794-797 (1997). The early studies involving oral immunization resulted in minimal and transient immune responses. Childers & Michalek, 1991; Childers et al., 1994; Childers & Michalek, "Controlled clinical study on responses to oral immunization with liposomes containing Streptococcus mutans glucosyltransferase," 76 Clin. Immunol. Immunopathol. S15 (1995). Therefore the more recent studies switched to other mucosal routes of immunization in an attempt to obtain improved salivary immune responses. One nasal immunization study was designed to test safety of IN immunization while comparing the immunogenicity of soluble versus liposomal C-GTF. This study involved twenty-one volunteers who were immunized by the IN route with 250 μg of C-GTF in liposomes or C-GTF alone (double blind), twice, seven days apart. Parotid saliva, nasal wash, and serum were collected prior to and at weekly intervals for 8 weeks following the first immunization for analysis of anti-C- GTF activity by ELISA. The levels of IgA anti-C-GTF activities increased in the nasal wash from both groups after immunization with a mean increase peak of 505% over 3 baseline samples on day 28. On day 28, the IgAl response in nasal wash for the liposomal C-GTF group, was significantly higher than that in the soluble C-GTF group. Salivary IgA anti-C- GTF responses were observed to a lesser extent. When the data was combined for both groups (since there was no difference between groups), there was a significant time effect, i.e., pre-immunization antibody levels were lower than post immunization levels (p<0.0001); and a mean increase peak of 73%, over 3 baseline samples on day 21. Significant serum IgA but not IgG responses were also noted. Significant differences between pre and post immunization samples were demonstrated for IgA in nasal wash, saliva, and serum when all subjects were grouped together (ρ<0.05). IgA responses were predominantly of the IgAl subclass. Childers et al., 1999. This study demonstrated the safety of the nasal route of immunization with C-GTF and showed that liposomal antigen was more effective than antigen alone at inducing secretory responses.

Another nasal immunization study was designed to compare the IN route of immunization to that of topical tonsillar immunization. This study involved four groups of five (or six) individuals immunized by the IN or topical tonsil route with soluble or liposomal C-GTF (125 μg). Parotid saliva, nasal wash, and serum were collected prior to and at weekly intervals for 8 weeks following the first immunization for analysis of anti-C-GTF activity by ELISA. S. mutans colonization was assessed by culturing saliva before and after immunization and a twice a day x 14 day chlorhexidine (0.12%) rinse. The immune responses determined by ELISA indicated that IN immunization was a better route of immunization than topical tonsil immunization in inducing significant nasal and salivary, but nonsignificant serum (IgA and IgG) responses. Within the IN immunization groups, liposomal antigen appeared more effective than soluble antigen; however, the difference was not statistically different for the small sample size. Salivary responses were less than the nasal wash responses observed. Recolonization of these subjects with S. mutans following prophylaxis and 14 day chlorhexidine rinse resulted in levels that were lower in the group of individuals immunized nasally with soluble C-GTF. These differences were not significant due to the variations observed between subjects and small sample number for the groups. Twelve of the subjects were recalled for 18 month follow-up samples and were willing to participate in a follow-up study to assess the effectiveness of a booster immunization. These individuals were originally immunized either by IN or tonsillar spray with soluble or liposomal C-GTF. Parotid saliva, nasal wash and serum were analyzed for persistence of immune responses by ELISA. Generally, all responses were lower than the peak responses, however, the mean IgAl anti-C-GTF antibody activity in nasal wash was significantly higher in the nasally immunized as compared to the tonsil-immunized groups. The conclusions of this study were that IN immunization at a lower dose than previously used (i.e., 125 μg vs. 250 μg C-GTF) was more effective than topical tonsillar immunization in inducing salivary and nasal (but not serum) responses that may have persisted for up to 18 months (nasal wash IgA). The ability to attribute some delay in recolonization with S. mutans following chlorhexidine mouth rinse was not possible due to high variation in oral samples as well as the lack of effectiveness of a 14 day mouth rinse with 0.12% chlorhexidine in eliminating S. mutans. Childers et al., 2002.

In another follow-up nasal immunization study, the twelve subjects that were followed up for 18 mo in the previous study plus twelve additional volunteers were recruited for a nasal immunization study where two groups of subjects received 62.5 μg of soluble or liposomal C-GTF twice, seven days apart. The objective of this study was to determine if subjects previously immunized 2 years earlier would show a memory response. The use of a lower dose of C-GTF would provide information about the dose response effect of the soluble or liposomal vaccine.

Parotid saliva, nasal wash and blood were collected weekly for two weeks prior to and for two months following immunization. A 3 -month post immunization sample was also collected. The nasal wash response was significant (mean peak in IgA anti-C-GTF 177% over baseline on day 21 in L-C-GTF group) but no difference was seen between groups. The salivary response was low (mean peak in IgA anti-C-GTF 43% over baseline in L-C-GTF group on day 35). The salivary response was found to be significant for the L-C-GTF (p=0.03), but not for the soluble C-GTF group. These findings indicate that at the lower dose, local (nasal wash) but not salivary response was maintained. Analysis of samples from the previously immunized subjects found that the responses in the previous IN immunized group was much higher (+ 60-200%, p<0.05) than that seen in the naϊve and previously topically tonsil immunized immunization groups. The salivary IgA anti-C-GTF response was lower in magnitude than the nasal wash response and no significant differences between groups were observed even though the DSf pre-immunized group was generally higher in magnitude. These data indicate that previous immunization may prime the immune system.

In summary, the results indicated that the higher doses of antigen produced higher (better) salivary immune responses although the magnitude is lower than nasal wash.

Specifically, the 125 μg dose of C-GTF induced a salivary immune response comparable to the higher (250 μg) dose tested. Li et al., "Intranasal immunization of humans with Streptococcus mutans antigens: low dose differentiates responses to soluble versus liposomal antigens," 18 Oral Microbiol. Immunol. 271-277 (2003). In a series of studies, healthy adult volunteers were immunized by the intranasal route with the recombinant saliva-binding region (SBR) of the S. mutans surface protein Agl/II with the adjuvant monophosphoryl lipid A (MPL) on days 0 and 14. A similar group of non- immunized individuals served as controls. All subjects received a dental prophylaxis on day 7 and treatment with the antibacterial mouthwash chlorhexidine daily for 14 days (Days 7- 21). Plaque and oral mouth rinse samples were collected from both groups of subjects prior to and following immunization and were plated on Mitis Salivarius and Gold's agar plates. The numbers of colony-forming units of mutans streptococci and of total streptococci were enumerated after incubation. Parotid and sublingual/submandibular saliva, nasal wash and serum samples were also collected. Saliva and nasal wash samples were assessed for IgA antibodies specific to Agl/II by ELISA. Serum samples were assessed for IgG and IgA antibody activity. A decrease in the percent & mutans/tot&l streptococci was seen in plaque and oral rinse samples in both groups of subjects following the dental prophylaxis and the chlorhexidine treatment. The percent S. mutans/total streptococci in plaque and oral rinse samples from the immunized subjects increased at a slower rate than seen in the non- immunized control subjects for up to six months. IgA anti-Agl/II immune responses were detected in saliva and nasal wash samples from the immunized, but not the non- immunized subjects. The kinetics of the responses differed in each secretion. Little or no serum IgG or IgA was detected. In using SBR as a mucosal vaccine in humans no complaints or adverse effects were reported.

An embodiment of the invention provides for a recombinant chimeric protein consisting of the two virulence determinants SBR and GLU (SBR-GLU) (Zhang et al., 2002) 5 useful for mucosal delivery in humans. The effectiveness of this construct in inducing mucosal and systemic immune responses to each virulence determinant following intranasal immunization was compared to that of each antigen alone or an equal mixture of SBR and GLU (SBR+GLU) in a mouse model. Further, the ability of antibodies induced to SBR-GLU in protection against S. mutatis infection in mice was also investigated. Immunization of mice

.0 with the chimeric protein SBR-GLU resulted in significantly enhanced (P < 0.001) levels of serum IgG anti-SBR antibody activity when compared to the SBR and SBR+GLU groups. The SBR-GLU immunized mice also demonstrated a significant (P < 0.05) increase in salivary and vaginal IgA antibody responses against SBR and GLU. Co-immunization with SBR and GLU resulted in significantly higher levels of serum IgG anti-SBR and anti-GLU

L 5 antibody activities than those seen in sham-immunized mice. No significant difference was seen in the serum IgG and salivary IgA anti-GLU or anti-SBR levels between the co- immunized group and the group immunized with GLU or SBR alone. Finally, a significant reduction (P<0.05) in S. mutans colonization was observed only in mice immunized with the SBR-GLU chimeric protein.

20 These results indicate, surprisingly, that the chimeric protein SBR-GLU significantly enhanced mouse mucosal immune responses to SBR and GLU and systemic immune responses to SBR. The ability of SBR-GLU in inducing responses effective in protection against colonization of S. mutans suggests its potential as a vaccine antigen for dental caries in other animals and humans. Furthermore, these results have provided evidence for the safe 5 use of this chimeric protein as a mucosal vaccine.

The SBR-GLU immunogenic protein may be prepared and delivered as a mucosal vaccine either alone, or either associated with an adjuvant or carrier or as part of an adjuvant or protein conjugate. Delivery by liposomes is provided for in greater detail below, and other systems include microparticles, virus-like particles, DNA vaccines, live recombinant vectors 0 such as Salmonella typhimurium, and possibly immune stimulating complexes ( ISCOMs). All of these systems are well-known by those of ordinary skill in the art, and may be practiced without undue experimentation. See, e.g., Michalek et al., "Antigen Delivery Systems I: Nonliving Microparticles, Liposome, and Immune Stimulating Complexes (ISCOMs)," in Mucosal Immunology (Mestecky et al., eds., Elsevier, 2005), incorporated herein by reference. Delivery of the immunogenic compositions of the present invention may be by parenteral, subcutaneous, intravaginal or intramuscular injection, or nasal, oral or rectal vaccination. The vaccine may also be delivered topically, including intranasal, upon the palatine tonsil, or delivery to the salivary glands.

For example, U.S. Patent No. 6,846,488 provides for methods of inducing immune responses by recombinant antigen-enterotoxin chimeric mucosal immunogens that contain the A2/B subunits of cholera toxin or heat-labile type II toxins. More specifically, this patent relates to a fusion protein in which an antigen (particularly SBR) is genetically coupled to the either an A2/B subunit-construct of cholera toxin, or a LT-II construct of heat-labile enterotoxin. Hence, in one embodiment of the invention, the SBR-GLU chimeric protein is either genetically or chemically conjugated to the toxoid carrier. In addition to the cholera and entero toxoid, other adjuvants suitable for use with the invention described herein include ricin toxoid, PorB proteins (see, e.g., U.S. Patent No. 6,613,336) and the like. Another promising protein-based mucosal adjuvant is the flagellin protein from S. typhirnurium. In mice, the flagellin protein (FIjB) when co-administered with the SBR antigen, induced higher anti-SBR antibody titers than did the SBR alone. FljB-exposed dendritic cells showed an increased expression of CD80 and CD86. FIjB augments mucoscal and immune responses (Ab and CD4+T-cell) when co-administered with SBR intranasally. FIjB stimulated and increased the expression of B7-1 and B7-2 costimulatory molecules on dendritic cells. In vivo data obtained from B7 knockout mice indicates that B7-2 is primarily responsible for FIjB to act as a mucosal adjuvant. Based on IgG subclass responses and CD4+T-cell cytokine production, FIjB enhances both ThI and Th2 associated immune responses. In an embodiment of the invention, the SBR-GLU protein is co-administered with the flagellin protein (FIjB) via, for example, the mucosal intranasal route.

Microparticles offer another alternative vaccine delivery system. Compounds useful for microencapsulation include starch, polyacrylamide, and co-polymers such as, for example, poly (lactide coglycolide) (PLG), polycaprolactone, (3-(triethoxysilyl)-propyl- treminated polydimethylsiloxane, or polymerized polysaccharide nanoparticles. For example, emulsions are prepared in which the protein antigen is entrapped within the PLG microparticles, with or without a co-stimulatory protein such as a toxoid. Immunization with a variety of antigens via nasal, intratracheal, or oral delivery has been shown to induce both serum and secretory antibody responses. Michelek et al., 2005. In an embodiment of the present invention, the SBR-GLU chimeric protein is delivered in a microparticle vaccine delivery system.

The chimeric protein of the instant invention may also be delivered as a DNA vaccine for in vivo expression of the immunogenic construct. For example, cationic microparticles may be used to deliver the DNA expression cassette in intranasal vaccination. Such systems have induced an immune response following, for example, intranasal delivery of vaccine comprising DNA encoding the HIV-I gag protein. Michalek et al., 2005. In an embodiment of the present invention, the SBR-GLU construct is delivered via a DNA expression cassette which is subsequently expressed in vivo. Another vaccine delivery system is often referred to as "virus-like particles", involving nonreplicating viral proteins that self-assemble into particulates in vitro. For example, U.S. Patent Application Publication No. 2004/0219164 describes an antigen presenting platform comprising self-assembling duck hepatitis core proteins genetically modified for antigen expression. Similar systems include replication defective viruses such as Sindbis replicons. Michalek et al., 2005. Hence, in another embodiment of the invention, the SBR-GLU chimeric protein is delivered as part of, or in association with, a virus-like particle.

Other adjuvants useful in the present invention include monophosphoryl lipid A, cholera toxoid, muramyl dipeptide, and ISCOMs. Although ISCOMs have not yet been used extensively in humans, this approach has been used in animals for year, and may serve as a potent vaccine adjuvant in humans. ISCOMs are complex structures composed of glycosides present in the adjuvant Quil A (derived from the bark of the Quillaja saponaria tree), cholesterol, the antigenic component, and in most cases a phospholipids such as phosphatidyl choline or phospatidylethanolamine. Often, the antigenic protein will readily self-assemble into the ISCOM structure. In some instances, the protein may be modified (e.g., genetically) to include a membrane insertion of anchor sequence, or be coupled to a hydrophobic carrier such as palmatic acid or lipopolysaccharides. Michalek, 2005. Thus, in one embodiment of the invention, the SBR-GLU construct is associated with an ISCOM.

The SBR-GLU chimeric protein of the present invention may also be expressed and delivered via a recombinant, colonizing bacteria, such as E. coli or Salmonella, or virus, such as polio. For example, S. sobrinus Spa A was expressed in an avirulent mutant S. typhimurium, and induced salivary IgA post oral immunization. In that study, a clone was also produced that co-expressed cholera toxoid CTA2/B. Hence, the live vector may also be combined with the expression of an adjuvant protein for increased immunogenicity. Attenuated mutants of S. typhi have been constructed to express hepatitis B antigen, and found to be safe and immunogenic when tested in humans. See, e.g., Hajishengallis & Michalek, "Current status of a mucosal vaccine against dental caries," 14 Oral Microbiol. Immunol. 1-20 (1999). Liposomal delivery is discussed in further detail in the Examples, below, thus in one embodiment of the invention, the SBR-GLU construct is delivered via liposomes. Liposomes also exhibit adjuvanicity in several ways. Liposomes protect the SBR-GLU antigen construct from acidic and enzymatic degradation in the intestine following oral vaccination. Additionally, particulate antigen is taken up more effectively than soluble antigen by M cells. Also, antigen depots are formed, in which antigen is maintained at local sites, minimizing systemic absorption.

The present invention is also directed to a method of inducing a B7-dependent immune response by administration of a recombinant immunogen expressed from a plasmid which comprises DNA sequence encoding a chimeric SBR-GLU antigen. The B7-dependent immune response includes induction of B7-2 expression on B cells or antigen presenting cells, B7-2-mediated co-stimulation of T cell proliferation, enhanced IgGl secretion, or induction of Th2 immune responses. In one aspect, the immune response results in the production of antibodies against the protein antigen that are present in saliva, intestinal secretions, respiratory secretions, genital secretions, tears, milk, or blood. In another aspect, the immune response includes the development of antigen-specific T cells in the circulation and tissues, development of cytotoxic T cells, or immunological tolerance to the protein antigen sequence.

In another aspect of the present invention, the SBR-GLU chimeric protein exhibits unique in vitro profiles regarding the toll-like receptors (TLR) dependent mediation of expression of costimulatory molecules B7-1 and B7-2, as well as MHC-II and certain cytokines. TLRs can be expressed by a variety of cells, including antigen-presenting cells such as monocytes/macrophage and dendritic cells. TRLs recognize pathogen-associated molecular patterns distinct from those of the host, but conserved among microbes, activating signal transduction pathways and inducing the production of certain cytokines. In particular, in vitro studies in mice indicate that the SBR-GLU chimeric protein signals through the TLR4 processing pathway. Stimulation of bone marrow-derived dendritic cells with SBR- GLU results in an increase in the expression of both BL7-1 and especially BL7-2, and of CD40. It also induces the production of TNF-α, IL-10, and IL-12p40. An aspect of the present invention provides for a method of inducing a TLR mediated immune response via exposure to the SBR-GLU protein.

An aspect of the present invention provides for the oral, tonsillar, and/or nasal administration of a vaccine that contains SBR-GLU to preadolescent and preschool children to elicit an immune response against mutans streptococci. According to this approach, the most advantageous route of immunization, resulting in the best salivary immune response, is provided for. The invention provides for both liposomal antigen and soluble antigen for immunization, and the most advantageous approach may be selected. An aspect of the present invention determines if mucosal immunization induces serum antibodies to human heart tissue. Another aspect of the invention provides for a safe, purified SBR-GLU vaccine for oral, tonsillar, and nasal administration to preadolescent children.

Without further elaboration, one skilled in the art having the benefit of the preceding description can utilize the present invention to the fullest extent. The following examples are illustrative only and do not limit the remainder of the disclosure in any way.

EXAMPLES

Example 1. Construction and purification of a chimeric protein consisting of two virulence antigens of S. mutans.

The plasmids ρET20(b)(+)-SBR and pET20b(+)-GLU, encoding the SBR of Agl/II and the GLU of GTF-I from S. mutans, respectively, were used to construct pET20b(+)-SBR- GLU (FIG.l). See also Zhang et al., 2002. The DNA segment encoding GLU was amplified by PCR with plasmid pET20b(+)-GLU. PCR primers were chosen with the help of the Oligo 4.03 primer analysis program (Nat'l Biosci., Inc., Plymouth, MN), and the appropriate restriction sites were introduced for subcloning (Xhol site at the 5' end of the upper and lower primers). The 0.9-kb gene segment encoding GLU was ligated in frame with the 3' end of the 1.2-kb segment encoding SBR in the pET20b(+)-SBR vector, resulting in the plasmid named pET20b(+)-SBR-GLU. This plasmid was introduced into E. coli BL21(DE3) by electroporation. The transformed colonies were selected in Luria-Bertani (LB) agar plates containing 50 μg/ml carbenicillin. The transformant was examined for the presence of a 5.8 kb plasmid by using the Wizard Miniprep DNA Purification System (Promega, Madison, WI). The presence of the insert was confirmed by Xhol digestion followed by gel electrophoresis. Recombinant SBR-GLU is a genetic chimeric protein with a molecular weight of 75 kDa. Briefly, Escherichia coli BL21(DE3) containing pET20b(4)-SBR-GLU is grown in LB broth containing 50 μg/ml carbenicillin at 30°C to mid-log phase and then induced with 0.36 mM isoprophyl-β-D-thiogalactopyranoside (IPTG) for 3 h. Following centrifugation, the cells 5 are suspended in binding buffer (0.5 M NaCl, 20 mM Tris-HCl [pH 7.9], 5 mM imidazole) and stored at -70⁰C. The cells are then thawed and sonicated, and the supernatant filtered through a 0.45 μm filter and loaded onto a His-Bind® resin column (Novagen, Madison, WI). After washing with binding buffer followed by washing buffer (same as binding with 60 mM imidazole), the SBR-GLU is eluted with 1 M imidazole. Following dialysis against PBS and 0 filtration through a 0.2 μm filter, the protein content is determined using bicinchoninic acid protein determination assay (Pierce, Rockford, IL) with bovine serum albumin as the standard. The purity of the protein preparation is determined by SDS-PAGE, Western blot using anti-GLU and anti-SBR antibody. The absence of LPS in the preparation is determined using the Limulus assay as recommended by the manufacturer (Biowhittaker, Walkersville,

[5 MD) and Western blot analysis using anti-LPS antibody. Both of these methods are very sensitive and are routinely used by those of ordinary skill in the art. The preparation may then be stored frozen (-70⁰C) until used.

The chimeric SBR-GLU protein construct is compared against SBR and GLU antigens delivered separately or together. These latter components are prepared and their

10 antigenicity determined as follows:

The SBR and GLU is purified for use in ELISA. SBR is purified from the soluble fraction of E. coli BL21(DE3) containing pET20b(+)-SBR by the same method used to purify SBR-GLU. GLU is purified under denaturing conditions from the inclusion bodies in the cytoplasmic fraction of E. coli BL21(DE3) containing pET20b(+)-GLU cells by known 5 methods. Briefly, following growth, the soluble proteins are recovered by resuspending the pelleted cells in binding buffer and stored for 1 h at -70⁰C. Following thawing, the cell-lysate is sonicated. The inclusion bodies containing GLU are recovered by centrifugation and solubilized in 6 M guanidine-HCl-0. IM NaH₂PO₄-ImM Tris-HCl (pH 8.0) by stirring at room temperature for 4 hours. The lysate is sonicated again, clarified by centrifugation and 0 loaded onto a His-Bind® resin column. The column-bound protein is washed with 8M urea- 0.1 M NaH₂PO₄-ImM Tris-HCl (pH 8.0), followed by 8 M urea-O.lM NaH₂PO₄-ImM Tris- HCl (pH 6.3). The protein is refolded by lowering the urea content of the refolding buffer (8 M urea, 0.5 M NaCl, 10 mM Tris-HCl, 20% glycerol [pH 7.4]) by 1 M steps and then eluting with 0.25 M imidazole hi refolding buffer without urea.

For Agl/II, MT8148, a S. mutans clinical isolate (S. Hamada, Osaka, Japan) is used to purify Agl/II for ELISA. Stock cultures of this strain are maintained frozen (-2O⁰C) in CDM containing glycerol. Purity of the culture is confirmed by streaking an inoculum from an overnight broth culture onto blood agar plates. Culture of S. mutans will be grown using chemically defined medium (van de Rijn & Kessler, "Growth characteristics of Group A streptococci in a new chemically defined medium," 27 Infect. Immun. 444-448 (1980)) (18 hr at 37°C). The cells are removed from the culture by centrifugation and 0.2 μm filtration using PLGC Pelicon Cassette system (Millipore Inc., Bedford, MA). The supernatant is then concentrated to approximately 1/20 original volume using the Pelicon Cassette system (10,000 MW cutoff). Proteins are precipitated from the concentrated culture supernatant with 60% saturated ammonium sulfate. Following centrifugation at 13,800 x g for 30 min, the pellet will be resuspended hi 0.1 M phosphate-buffered saline (PBS, pH 7.4) and dialyzed extensively against PBS at 4°C to remove ammonium sulfate. To purify Agl/II, the crude preparation is chromatographed on a DEAE Fast Flow column (Amersham-Pharmacia Biotech, Piscataway, NJ) with a step gradient of 0.1M NaCl. Purity will be determined by SDS-PAGE (7.5% acrylamide gels) silver stain and Western blot analysis using an antibody against Agl/II. Regarding GTF for this Example, GS-5, a S. mutans clinical isolate (F. Macrina,

Virginia Commonwealth University, Richmond, VA) is used to purify GTF. Identical methods as used for Agl/II will be used to obtain a crude culture supernatant (i.e., Pelicon system concentration, ammonium sulfate precipitation and dialysis). GTF is further purified using a modification of procedures of Taubman et al., "Immune properties of glucosyltransferases from S. sobrinus," 17 J. Oral Pathol. 466-470 (1988). The concentrated supernatant is applied to a Sephadex G-100 (Amersham-Pharmacia) with 3 M guanidine HCl as the eluting solvent. This GTF-rich pool is then filtered on a column of Sepharose 4B-CL (Amersham-Pharmacia) with 6 M guanidine HCl for elution. This method has been shown to obtain a mixture of GTF isozymes including GTF-I and GTF-S, but essentially free of other proteins. GTF will be subjected to SDS-PAGE (7.5% acrylamide gels) analyses, and the purity and enzymatic activity of GTF will be determined by Silver stain and Periodic Acid Schiff (PAS) stain following incubation of the gel with sucrose, using known methods. Regarding studies of heart sarcolemma, fresh human heart is obtained from any number of sources, such as UAB Tissue Procurement for preparation of sarcolemma protein to be used to coat ELISA. Sarcolemma preparations are made by known methods. Briefly, 10 grams of fresh ventricular heart muscle from autopsy of a healthy adult (i.e., accident victim) will be homogenized in 0.05 M CaCl₂ for 5 minutes on ice. Following centrifugation at 14,000 x g, the pellet will be washed in saline and then lysed using sterile water. Following treatment with DNase and RNase, sarcolemma obtained will be lyophilized until used for ELISA. Microtiter plates will be coated with 50 μl per well of 5 μg/ml sarcolemma (see below for general ELISA method). Human cardiac myosin is purified from fresh cadaver heart obtained from UAB

Tissue Procurement according to known procedures. Briefly, tissue is homogenized and extracted in buffer, with purified myosin obtained after three successive precipitation/solubilization cycles in low and high salt solutions to sequentially remove heart muscle residue, actin and actomyosin. The purity of the myosin is determined by SDS-PAGE and will be identified with a myosin-specific monoclonal antibody by Western blot (Sigma Chem. Co.).

Regarding antibody reagents, polyclonal reagents to human Ig are obtained commercially. Affinity purified F(ab')₂ goat anti-human IgA, and IgG is obtained from Jackson Imniuno Research Lab, Inc. (Avondale, PA); biotin-labeled F(ab')2 fragments of goat IgG antibodies against human IgA, IgG from Biosource (Burlingame, CA); peroxidase- labeled goat anti-mouse Ig (for monoclonal detection) from Southern Biotechnology Associates (Birmingham, AL). Mouse monoclonal antibodies specific for IgAl and IgA2 (Southern Biotech. Associs.) are available in a purified or peroxidase-labeled form and have been extensively used in our laboratories. See Childers et al., 1994. The specificity of the human reagents for use in ELISA has been confirmed using purified colostral S-IgA and myeloma proteins as standard.

Proteins for standards include human myeloma plgAl, mlgAl, pIgA2, and colostral S-IgA, which have been purified by procedures previously by methods well known in the art. For ELISA analyses, optimal concentrations of SBR, GLU, Agl/II and GTF (or sarcolemma/myosin) are diluted in 0.1 M carbonate buffer (pH 9.6) for overnight incubation in 96-well polyvinyl chloride microtiter plates (Dynatech Laboratories, Alexandria, VA) at 37°C. For human total immunoglobulin determinations, antiserums to IgA or IgG are diluted in PBS to maintain Fab binding ability. Nonspecific binding sites are blocked with 5% fetal calf serum (Flow Laboratories, McLean, VA) in PBS-Tw. After 1 hr of blocking, duplicates of four 2-fold dilutions of specimens and six dilutions of a serum pool (i.e., human used as standard) or colostral IgA (i.e., saliva IgA standard) are added to individual wells. Depending on the specific analysis, appropriate dilutions of biotin- conjugated goat antiserum to human IgA or IgG, mouse monoclonal anti-human IgAl or IgA2 are added to appropriate wells. Wells are then developed with streptavidin-alkaline phosphatase conjugate followed by phosphatase substrate Sigma 104 tablets in diethanolamine buffer. Color development (O.D.) is recorded at 405 nm using a ELISA plate reader (Vmax, Molecular Devices Corp., Menlo Park, CA). A 4-parameter curve fitting program (Softmax, Molecular Devices Corp.) is used to construct reference curves for each ELISA plate from O.D. readings of a standard serum assigned ng/ml. O.D. readings of the sample dilutions are compared to this curve to obtain ng values that are multiplied by the dilution factor to determine the ng/ml of antibody activity. For each human specimen, results obtained from baseline samples are averaged for comparison of pre- and post-immunization ng/ml values. Absolute levels of total immunoglobulin (i.e., μg/ml) for human specimens will be determined by construction of standard curves using Moni-Trol (a blood-based quality control product used as standard, American Hospital Supply Corp., Miami, FL) for serum and purified colostral IgA for saliva. For human studies, secretory immune responses will be reported as the peak percent increase of "corrected specific antibody activity" compared to mean baseline "corrected specific antibody activity."

Example 2. Preparation of liposomes and quality control procedures.

The chimeric protein may be used for mucosal vaccination as a soluble protein or in association with liposomes. Regarding liposomal preparations, the components used for production of liposomes are dipalmitoyl phosphatidylcholine (DPPC; Avanti Polar Lipids,

Alabaster, AL), cholesterol (Avanti), and dicetylphosphate (DCP; Sigma Chemical Company, St. Louis, MO). Liposomes are made by dissolving DPPC, cholesterol, and DCP in chloroform at a molar ratio of 16:7:1, respectively. A lipid monolayer is formed in a round bottom flask in an N₂ atmosphere by rotary evaporation, then vacuum drying. A heterogeneous preparation of liposomes are formed when an aqueous solution (Hanks' balanced salt solution, HBSS; GIBCO, Grand Island, NY, containing 1.75% sodium bicarbonate for oral vaccine, PBS for nasal and tonsillar vaccines) of SBR-GLU is added to the lipid monolayer and mixed at 6O⁰C giving a total lipid concentration of 2 mg/ml. The resulting heterogeneous liposomes are made more homogeneous SUL by bath sonication (FS- 14, Fisher Scientific, Norcross, GA) for 20 minutes, then filtered with a 5 μm filter (Acrodisc, Gelman Scientific Co., Ann Arbor, MI).

Small unilamellar liposomes (SUL) are produced by extruding large multilamellar liposomes through a 100 nm pore membrane (LiposoFast, Avestin, Inc., Ottawa, Canada). These sterile liposome preparations are characterized by flow cytometry (see below). Extruded SUL are frozen and dehydrated following addition of 250 mM trehalose (cryoprotectant, Sigma) by quick freezing in glass test tube using dry ice and alcohol, then dehydrated by lyophilization. The resulting dehydrated liposomes are rehydrated with the original volume of sterile distilled water.

Liposome preparations are characterized for relative size and homogeneity by flow cytometry (FACStar™, Becton Dickinson, Mountain View, CA), as previously described. Childers et al., "Characterization of liposome suspensions by flow cytometry," 119 J. Immunol. Methods 135-143 (1989). Polystyrene 0.130 μm beads (Fluoresbrite beads, Polysci. Inc., Warrington, PA) are used for FACStar™ calibration and standardization to characterize liposome preparations. Sonicated liposomes generally have 50-60% of particles in the submicron range, while filtered (5 μm) liposomes consistently have greater than 75% in the submicron range. An acceptable level of percent decrease in submicron particle counts has been established by comparison of this data to transmission electron microscopy. Id. Optionally, for oral administration, enteric coating is done using cellulose acetate phthalate dissolved in water, acetone, and triethyl citrate by known methods. See, e.g., Czerkinsky et al., 1987.

Note that among the other procedures for antigen preparation and vaccine testing which have already been described, the FDA requires the following preclinical standardization and safety tests be conducted prior to beginning clinical studies: Each lot of SBR-GLU must be shown to be immunogenic in a suitable animal model. To demonstrate immunogenicity, 6 BALB/c mice are injected intraperitoneally with 50 μg of SBR-GLU. Pre (day 0) and post (day 14) immunization blood samples are collected and serum analyzed by ELISA (see method below). Acceptable immunogenicity is typically demonstrated by a mean increase of pre to post immunization serum (1 : 1000 dilution) from less than 0.1 to greater than 1.0 optical density units. Sterility testing of the final products prior to human administration is conducted by submitting to an infectious disease laboratory such as the Division of Infectious Diseases Laboratory at UAB Department of Medicine. Their procedures are in compliance with the 21 CF .R. 610.12 guidelines, which involves growing in thioglycolate media for a period of 14 days. To evaluate the biological burden of the contaminants, 0.1 ml samples are plated onto blood agar incubated aerobically and anaerobically at 37°C for 48 hours. Acceptable levels of biologic burden are less than 400 CFU/ml of preparation for oral immunization studies. The FDA has recommended, however, that preparations for nasal use should be minimally contaminated and bacterial contamination should be typed and speciated to document that the organisms are not associated with pathogens. An infectious disease laboratory is fully capable of speciating organisms. Regarding general safety of product in final form, two BALB/c mice (body weight less than 22 grams) for each preparation are injected intraperitoneally with 0.5 ml of product that is to be used to immunize humans. In a similar approach, two guinea pigs less than 400 gram body weight each will be injected intraperitoneally with 5.0 ml of product for human use. The animals are monitored for adverse reaction and weight loss for a period of one week. If required, the results of the above tests as well as other preclinical data and the proposed protocol are submitted to the FDA prior to beginning each study. In such instances, within 30 days the FDA in turn will respond with any concerns and recommendations. Unless concerns are addressed to the satisfaction of the FDA, studies are not allowed to begin.

Generally, the safety of liposome preparations is a function of their components and the physical nature of the vesicles formed. The components used in liposome preparations are safe for use in humans, widely used by others, and are described below. Components:

1. Dipalmitoylphosphatidylcholine (DPPC) is a naturally occurring lecithin substance found in many animal and plant sources such as egg yolk and soybean oil. It is a normal component of cell membranes and it is readily biodegradable. DPPC itself is a poor antigen, and therefore does not elicit allergic reactions. This substance is the main ingredient of the microspheres that form in aqueous solutions by virtue of the bilayer alignment of hydrophobic and hydrophilic portions of the DPPC molecules.

2. Cholesterol is a naturally occurring essential fat that is found in virtually all- vertebrate animals. Although high levels of this substance may cause arteriosclerosis, the amounts that will be administered for this study is minute (0.45 mg/dose for oral) compared to that in a normal human diet. Cholesterol is added to increase the stability of the liposomes. 3. Dicetylphosphate is added to create negatively charged surfaces on the liposomes. The molecular nature of the surface charge on liposomes is an important determinant feature for the adjuvant properties of these microparticles. Small (50-100 nm), unilamellar, negatively charged liposomes have been reported to cause macrophage activation and 5 pinocytosis for molecular processing of antigens. This property is also important for the selective uptake of the particles by host tissue.

Extensive studies have been conducted in animals and many pilot studies in adult volunteers to determine the effectiveness of oral as well as nasal and tonsillar immunizations consisting of antigen in liposomes. In these studies, no adverse effects were observed. The .0 studies in preadolescent and preschool aged children reflect experience in animal and human studies. Furthermore, the accumulating evidence supports the contention that liposomal preparations are safe for human mucosal administration.

Example 3. The effect of age on the immune responses of children following mucosal

L 5 immunization with recombinant SBR-GLU chimeric protein.

The present invention provides for the immunogenicity of a recombinant MS antigen as a mucosal vaccine when given by oral, nasal, or topical tonsillar route to adults, elderly adults and children. This Example addresses the immunogenicity of a recombinant MS antigen as a mucosal vaccine when given by oral, nasal, or topical tonsillar route to different

20 age groups in children.

The basic assessments of interest essentially require identifying correlates of the salivary IgA anti-SBR, anti-GLU, anti-Agl/II, and anti-GTF antibody activity based on group assignment. Thus, it is proposed to perform various correlation analyses. These will include the correlation of various demographic, cultural, and socioeconomic factors as well as 5 levels/colonization with mutans streptococci with the level of salivary IgA anti-SBR, anti- GLU, anti-Agl/II, and anti-GTF antibody activity. In addition to the usual forms of correlation analyses (Pearson correlation, Spearman nonparametric methods as well as contingency tables for testing for associations), the data points in time may be able to utilize to develop a path analyses. Briefly, the hypotheses previously described will be reviewed 0 with the appropriate directional and increased acceleration of response considered, and terms for the intervention, its timing, and possibly the adherence will be included. In the plan for data analyses, it is proposed to investigate the effects of the numerous factors as hypothesized above as these relate to outcome factors such as antibodies. The rationales for these measurements suggest expected directions of effects. These analyses are addressed by regression analyses techniques (general linear models and/or logistic regression). The SAS computer package will be utilized to perform these analyses. In summary, the approach of intent-to-treat, two tailed Tests (α = 0.05), ANOVA, Cochran-Analyses Mantel-Haenszel statistic, non-parametric methods and logistic regression as appropriate will be used.

A modified 3x2 factorial design is used with 3 vaccine routes and two delivery systems (liposomal or soluble SBR-GLU). Preadolescent (age 10-12) and preschool (age 5-6) children of consenting parents are recruited from the pediatric dental clinics or hospitals, such as the Children's Hospital and University of Alabama School of Dentistry. Based on results from studies in adults, group sizes of 10 subjects should be sufficient to yield meaningful results (see sample size justification below). The design proposed for this study identifies the mucosal response elicited by an optimal vaccine regimen by testing all combinations of the factors used in previous adult studies. Thus, the proposed modified factorial design allows for evaluation of "all" main effects and interactions. This approach is well known in the art. See, e.g., Federer, Experimental Design: Theory and Application (Macmillan Co., New York, 1963). Additionally, it is likely this group size range will be more likely to discriminate differences between variables (i.e., is a conservative estimate of sample size because the mucosal immune system in children is potentially more naive and therefore, more active in this age group than in adults). A factorial design will include route of immunization (oral, nasal, and tonsillar) x antigen form (soluble antigen and liposomal antigen). The design is designated "modified" because oral immunization requires higher dose (i.e., 500μg versus 125 μg for nasal and tonsillar) and a different protocol (seven consecutive days of immunization versus two doses for nasal and tonsillar), but still represents the route differences. The rationale for the antigen, dose, and immunization protocols are novel and non- obvious, particularly in the generalizability to children, but yet are based on previous studies {see, e.g., Childers et al. 1991 and 1994; Smith & Taubman, 1990 and 1987), as mandated by NIH. Blood and saliva are collected for analysis bi-weekly or monthly throughout the study: prior to, during, and following immunization. Pre-samples of saliva and blood are collected prior to immunization to establish the baseline antibody activity as well as the variation in natural antibody levels over short periods of time. Saliva and serum samples are analyzed by ELISA to determine changes in anti-SBR, anti-GLU, anti-Agl/II and anti-GTF antibody activity. Although the use of the recombinant antigen SBR-GLU is not expected to induce antibodies that cross-react with human heart tissues, because of early concerns with streptococcal vaccines, serum samples are monitored for the presence of cross-reacting antibodies (i.e., induction of IgG anti-sarcolemma and anti-myosin antibody activity). The appropriate safety and data monitoring boards are apprised routinely of studies and the safety of children is monitored internally, externally, by, for example, institutional review boards. In order to standardize all aspects of the specific studies, one large lot of purified SBR-GLU is obtained and used. Monitoring of the stability of SBR-GLU by SDS- PAGE and Western blot analysis continues throughout studies. The stability of dehydrated liposomal preparations has been established {see, e.g., Childers et al., 1997). Therefore, one large batch of SBR-GLU and liposomal SBR-GLU (L-SBR-GLU) may be used in an entire study, and may be prepared and tested for analysis of as required by the FDA prior to each smaller component study. In an aspect of the present invention, the SBR-GLU can be stably stored for at least three years. In the event that stability of SBR-GLU becomes an issue, a new batch is prepared and checked for its similarity to the initial preparation prior to initiation of a study.

The total number of subjects for the factorial design is 120: 10 (subjects/group) x 3 (test routes) x 2 (antigen forms) x 2 (age groups). Subjects are randomly assigned to experimental groups after balancing for sex and race. This sample size is determined using fixed effects analysis of variance power analysis by known methods. See, e.g., Kraemer & Thiemann, How Many Subjects? Statistical Power in Analysis in Research (SAGE Pubs. Inc., Newbury Park, CA, 1989). Ten subjects/group (12 groups, 120 subjects) were estimated to be the sample size necessary assuming a relative effect size of 0.4 and a type I error rate of 0.05 which results in a power estimate of greater than 0.96 for all factors and combinations. Although a relatively high effect could be overly optimistic, because this study identifies a factor combination that greatly surpasses others, it is not unreasonable to set the effect size high. Nonetheless, even with an effect size of 0.3, power levels at this sample size are approximately 0.8 or greater. Table I below represents the results using the geometric mean. A two-way analysis of variance with a 0.05 significance level will have 99% power to detect a variance among the 3 Factor A means of 0.011, has 99% power to detect a variance among the 2 Factor B means of 0.001, has 89% power to detect an interaction among the 3. Table I: Geometric Mean hypothesized based on Adult Studies.

Factor A levels and the 2 Factor B levels of < 0.001, assuming that the common standard deviation is 0.050, when the sample size in each group is 10. Thus, for each age group there is sufficient power to assess the test routes and antigen forms. Further, while one would expect the result to be similar by age - there is sufficient power to assess age or maturation differences.

Healthy volunteers are recruited in the dental clinics, e.g., at the School of Dentistry and Children's Hospital. Such clinics are prevention oriented and may treat approximately 1000 patients per month. Of this number, the number of children in the age ranges from which children are recruited are estimated to be approximately 500 per year each for children age 5-6 and 10-11 years. Therefore, an adequate number of patients is available for recruitment (i.e., approximately 60 per year). These children and parents are provided written informed consent for participation and HIPAA compliance. They may receive various incentives to reimburse them for their time and travel expenses and compensate for inconvenience of multiple visits during the active part of the study.

Criteria for selection of volunteers for this study may include age 10-11 (or 5-6) years old; parental consent; and plan to stay in the area for at least 12 months. All subjects must be free of systemic disease such as birth defects, bleeding disorders, tonsillectomy, kidney disorders, endocrinal disorders, bone disorders, asthma and allergy, cancer, HIV, and epilepsy.

Regarding sample collection and analysis, unstimulated parotid saliva is collected using plastic intraoral suction cups (Schaefer cups) positioned over the orifice of both parotid ducts. Saliva is then clarified by centrifugation at 5,000 x g for 2 min, aliquoted and stored at -70⁰C until used for ELISA. Saliva data obtained for relative IgA antibody activity (ng/ml) will be normalized by dividing the level of antibody activity by the total level of IgA in each secretion (corrected specific activity = ng anti-MS (i.e., anti-SBR, anti-GLU, anti-Agl/II & anti-GTF)/μg total IgA). This method of normalization assumes that specific antibody isotype concentration and total IgA concentration levels vary linearly and the ratio of these concentrations will remain constant for a given specific antibody activity level, independent of flow rate and other collection variables: i. levels of S-IgA, S-IgAl and S-IgA2 antibody specific for SBR, GLU, Agl/II, and GTF; and ii. total IgA, IgAl and IgA2 levels.

Blood is collected from a finger stick into a microvette tube with clotting activator (Sarstedt, Numbrecht, Germany). Following centrifugation, serum is collected, aliquoted and stored. Serum data obtained will be reported as relative antibody activity (ng/ml): i. levels of IgA and IgG antibody specific for SBR, GLU, Agl/II, and GTF; and ii. levels of IgG anti- human sarcolemma and myosin preparations.

Laboratory personnel are blind as to group assignments and therefore are provided with samples labeled with a code to identify the sample type, sample number and subject number only. All data is entered into the database with this identifying code. Mixed model analysis of variance (with α = 0.05) are used to compare average levels among the test groups and to ascertain group differences in patterns of responses across the repeated measurements on the same subject. When the raw data exhibit distributional skewness and evidence of non normality, appropriate transformations, e.g., logarithms, will be used to improve normality and to decrease heteroscedasticity and improve power.

Regarding the immunization protocol, the initial study is a 3 x 2 factorial study in the preadolescent children. This is followed by a similarly designed study with preschool children yielding the 3 x 2 x 2 set of studies. This rationale for staged designs by age is to insure safety in children, using older children first and then moving to the younger children. For seven days following immunization, subjects complete a daily diary with specific questions about any adverse events. Space is provided in this diary for comments as to the duration, severity, and timing of any adverse symptoms that may be associated with immunization. Regarding the route of administration (oral, nasal, and tonsillar immunization), dehydrated vaccine preparations are obtained by lyophilization as described in Example 2. After dehydrating a known quantity of antigen, the appropriate antigen dosage will be distributed into gelatin capsules for enteric coating for oral immunization. Alternatively, for nasal and tonsillar immunization, immunogen is distributed into sterile tubes according to weight and then hydrated with the appropriate volume of sterile distilled water just prior to immunization. The aqueous vaccine suspension is distributed into sterile vials for spray using bi-dose metered nasal spray applicator (Pfeiffer, Princeton, NJ). Based on previous studies in adults, topical immunizations twice (on day 0 and 7) and for oral immunization seven consecutive days were effective. The dose of SBR-GLU to be used for topical application will be 125 μg/dose because previous studies in adults indicated this formulation/dosage was immunogenic after nasal immunization. Childers et al., 1994. The oral antigen (soluble or liposomal) will be given in enteric coated capsules with 500 μg/dose as the dose as previously used and reported in adult studies. Pretreatment saliva and serum samples for ELISA are collected at day -14 and -7, a baseline sample at time 0 and then samples at days 21, 35, 56 and 90. The general study design for sample collection and immunization is provided in FIG. 4.

These studies show that the SBR-GLU vaccine is immunogenic and safe in preadolescent children, and therefore support study in preschool children. Based on previous adult studies as well as the fact that the vaccine consists of no foreign components (i.e., MS are indigenous bacteria), no reactions to the immunizations are expected. From previous studies and those of others it is not anticipated that serum IgG anti-sarcolemma or myosin antibody activity will be detected. If such antibody activity is detected and correlates with immunization, human studies are discontinued until the DSMB, FDA, and IRB are consulted for a decision as to whether limited (i.e., immunization routes or formulations that do not result in any serum response) immunization should continue.

The information that obtained from this Example includes a determination of the relative immunogenicity of six factors in two age groups of children. The expected results are shown in Table I above. This table is based on previous studies in animals and humans; no immune response is expected to orally administered soluble SBR-GLU, therefore, this group will probably serve as a control group. Note that although no response was detected following tonsillar immunization in adults, children have more active tonsillar tissue, which explains the expectation for positive response. The factorial design of this Example provides an indication of the route/delivery system and age group that will be a good choice for the study presented in Example 5 below. Previous studies in adult subjects indicated that nasal immunization induced higher and more persistent responses than oral and topical tonsillar routes. As previously discussed, the modified factorial design using more complete experimental units allows mixed model analyses to more efficiently evaluate main effects and interactions which will likely result in clear indication of best factor combination. It may also be important to evaluate the predominance of an IgA subclass response in saliva as was observed in oral immunization studies in adults. These analyses provide insight into a better understanding of the human immune system in children and provide indications of potential immunization approaches for other vaccines. The data from this series of studies also provides indicia of differences between mucosal immune responses of preadolescent and preschool children. Serum analysis may be important to evaluate for completeness in characterization of immune responses and to screen for any heart cross-reactive 5 antibody activity.

Example 4. Establishment of mutans streptococci on newly erupting permanent molar teeth of children.

This study determines how newly erupting molar teeth become colonized with mutans

[0 streptococci (MS). This study determines factors related to colonization of permanent first and second molar teeth in preschool and preadolescent age children, respectively, and estimates the time course of colonization in these age groups. More specifically, this approach answers the questions: how does a newly erupting permanent molar tooth become colonized with MS?; how can colonization of a newly erupted tooth be quantified?; what is

L 5 the timing of colonization of a newly erupted molar tooth?; and what factors are related to oral colonization of mutans streptococci on newly erupted permanent molar teeth in children? This approach determines the characteristics of the establishment of MS by two cross- sectional studies in preadolescent (age 11-13) and preschool (age 5-6) children, respectively. Assenting children of consenting parents, are recruited from pediatric dental clinics and a

20 detailed baseline questionnaire examined. Children meeting entrance criteria are enrolled, the stage of eruption of "pertinent" permanent molar teeth is recorded and oral samples collected for determination of biological factors related to the characteristics of colonization with MS (see below). In order to maximize acquisition of pertinent data, children are recruited with more than one molar in the process of erupting. Although, longitudinal data are important to

25 determine the timing of establishment of MS on newly erupted teeth, our study design takes advantage of four newly erupting teeth that can be sampled in that each subject and each tooth maybe at a different stage of development.

The amount of data collected on each subject provides virtual longitudinal data controlling for many factors, because this is a within-person study, using the estimated time 0 within the mouth for each molar from this cross-sectional survey. Therefore, the timing of sample collection (i.e., subject selection) is optimized by seeking subjects with two to four "relevant" teeth in the process of eruption. A method of staging of tooth eruption is used to characterize the timing of colonization (i.e., number of cusp tips-only exposed, one-third, one-half, two-thirds, fully erupted). Colonization is defined as plaque MS/total streptococci within 10% of that found on fully erupted molars (i.e., permanent 1st molars and primary 2nd molars for preadolescent and preschool age groups, respectively). In this regard, if the plaque of a fully erupted tooth has 5% MS/total streptococci, then colonization of a newly erupting tooth will be defined by achieving at least 4.5% MS/total streptococci from its plaque. A 0.05 one-sided Fisher's z test of the null hypothesis that the Pearson correlation coefficient = 0.3 or higher, has 80% power to detect a correlation coefficient of 0.30 when the sample size is 68. Hence, enrolling 70 children is adequate. The power is actually much larger because there are repeated measures over time on each child which can be related to the purported correlates using generalized linear models (PROC MIXED in SAS) and the imputed longitudinal nature of the data (time of each tooth's exposure is estimated within each child at the cross- sectional exam).

Group sizes of 70 subjects per age group should be sufficient to yield meaningful results, although perhaps not always statistically significant differences for all the variables tested, but for correlations that are likely to be biologically important. Because only one sample collection period is required for each child, recruitment of at least 70 subjects each year for this aim is not be a problem if approximately 1000 children of the two age groups are seen in the participating clinics each year.

Oral rinse and plaque samples are collected from children that meet the entrance criteria. Plaque samples consist of "adjacent" fully erupted and all partially erupted molar teeth. Oral rinse and plaque samples provide data to assess the presence of MS for correlations with stage of eruption compared to that of fully erupted molar teeth and salivary levels of MS.

Possible variables and end points include: Demographic and ID variables: Name, DOB, gender, race; Level of MS in saliva; Level of MS in plaque of adjacent molar teeth; Level of MS in plaque of erupting molar teeth; Salivary IgA anti-MS data from ELISA; Diet analysis for assessment of caries risk factors; Caries risk assessment (i.e., low, moderate, high risk); Caries activity (DMFS); Staging and timing (i.e., from subject self report) of eruption of each permanent molar tooth. Intermediate and "end point" variables: relative assessment timing for MS to colonize erupting molar teeth based on above data (i.e., MS level of adjacent molar teeth). The eruption staging/timing will be used as a covariate in the analysis by recording it and adjusting for its effect. This will facilitate more direct comparison of eruption time and time to colonization data. More specifically, regarding recruitment, selection, and specimen collection, this is an epidemiological cross-sectional study with consecutive recruitment of eligible children. Selection criteria for this study are the same as in Example 3 from clinic patients who present for preventive care (i.e., new patient visits, recall, and sealants). An additional requirement is that at least one "pertinent" molar has erupted. Due to the cross-sectional nature of this study, samples are collected only once (at the appointment when subject is recruited, i.e., before any preventive treatment begins).

Subjects are requested to rinse with 10 ml of sterile saline for 30 seconds and then expectorate into a sterile tube for salivary MS and total streptococci determinations (see below). Plaque samples for bacteriologic analysis will be obtained using a sterile toothpick and collecting plaque from the occlusal, buccal, and lingual surfaces of representative molar teeth present and each newly erupting permanent 2nd (or 1st) molars. In this regard, 4 individual plaque samples will be collected from "erupted" molars (i.e., permanent 1st molars or primary 2nd molars for preadolescent and preschool groups, respectively) for comparison to "erupting" molars. Plaque will be placed into sterile saline (ImI) for immediate processing (see below). Unstimulated parotid saliva will be collected and processed for ELISA as in Example 3.

Regarding sample analysis, for the oral sample for enumeration of MS per total streptococci, all saline mouth rinse samples should be transported to the microbiology laboratory on ice for processing within 2 hr of collection. Samples are mixed gently and dispersed sonically for 30 sec within a custom-adapted cup horn sonicator so that the vial can remain sealed and unexposed to atmosphere. Following dispersement, samples are plated using the Autoplate™ (Spiral Biotech, Bethesda, MD). Plates will be incubated at 37⁰C in an anaerobic atmosphere within a MACs anaerobic chamber (Microbiology Int'l, Gaithersburg, VA) for 2 days. Total cultivable streptococci are determined by counting colonies on Mitis Salivarius agar (duplicate plates from 10"¹ dilution). Streptococci will be initially identified based on colony morphology. MS are identified and counted following growth on Gold's selective media (undiluted, in duplicate). Colonies are identified and counted based on morphology. A limited number of sample colonies are studied by biochemical assimilation characteristics (Minitek, Becton Dickinson Co., Cockeysville, MD) to confirm mutans streptococci. A plater grid will be used to identify the optimal counting areas for each plate and the number of colonies counted. The number of colonies counted/plate will then be converted to colony-forming units (CFU) per ml saline rinse. The percent MS is determined by dividing the MS count (from Gold's medium) by the total streptococci (Mitis Salivarius medium).

For the plaque samples used in determining the time to colonization of permanent 2nd and 1st molars with MS, samples transferred to saline tubes are mixed gently and then sonically dispersed for 30 sec with sonicator (as above). The samples will be plated in duplicate using the Autoplate™ onto Gold's and Mitis Salivarius plates and incubated as above. Select colonies will be confirmed to be mutans streptococci by biochemical assimilation. Data will record the time from initial emergence of tooth to initial detection of MS on any 2nd (or 1st) molar as well as the percent MS (when colonization occurs). Colonization will be defined as the relative proportion of MS per total streptococci of the newly erupted molar which is similar (i.e., within 10% of the MS/total streptococci proportion) to other already existing molar teeth (i.e., permanent 1st molars or primary 2nd molars, for preadolescent and preschool children, respectively.

Salivary anti-MS IgA levels are determined by ELISA as described in Example 3. Laboratory personnel will be blind as to group and tooth assignments and therefore will be provided with samples labeled with a code to identify the sample type, sample number and subject number only. All data will be entered into the database with this identifying code. Longitudinal data models (with α = 0.05) will be used to compare average levels among the teeth, time in the mouth exposure variables and covariates to ascertain group differences in patterns of responses across the repeated measurements on the same subject. When the raw data exhibit distributional skewness and evidence of non normality, appropriate transformations, e.g., logarithms, will be used to improve normality and to decrease heteroscedasticity.

The data collected from oral samples for assessing MS and total streptococci colonization as well as for the assessment of the relative timing of initial colonization with MS of newly erupted permanent molars are evaluated for correlation with factors such as diet, caries risk assessment, salivary IgA anti-MS, level of salivary MS and proportion of MS per total streptococci on adjacent molar teeth (i.e., permanent first molar and primary second molars in preadolescent and preschool aged children, respectively). The plaque sample collection provides preliminary data for the relative time of colonization with MS that can be expected in the Example 5 studies, and therefore provides information to the research plan on the timing for the immunization to determine the effect of immune response to SBR-GLU on MS colonization of newly erupted molars. In this regard, if the findings from this Example indicates an obvious association with the timing of MS colonization of new teeth with the level of MS in the oral cavity (i.e., colonization is immediate with high level of MS), then Example 5 may need to include a protocol to decrease the level of MS in the oral cavity at the time of immunization (i.e., prophylaxis and chlorhexidine mouth rinse, see below). It may also apply to one age group and not the other. The use of prophylaxis and chlorhexidine rinse has previously been used and although not ideal, there is a consistent decrease that can be observed in MS levels. However, these levels, generally return over time to the baseline numbers. Nonetheless, this study design (i.e., including antimicrobial treatment) at least would be as good as that done previously in adult studies which indicated that immunization may correspond with a delayed recolonization with MS. Note that obtaining data of how colonization of newly erupted permanent molar teeth is new information and is beneficial to our understanding of biofilm development of newly erupted teeth in addition to providing information for the study design in Example 5.

Example 5 Determine the effect of salivary anti-SBR-GLU immune responses on MS colonization of newly erupting permanent molar teeth.

A relevant follow-up to Example 3 determines if salivary IgA anti-MS induced in Example 3 has functional activity, particularly the effect of salivary immune responses on colonization of newly erupted permanent molar teeth. In addition to considering the immunoresponsiveness from Example 3 studies, the data collected on general eruption patterns of new permanent molars and information on factors related to time to colonization is used to design the specific protocol of immunization and collection of samples for analysis of immune response and how immune response correlates with MS colonization.

In this study, the longitudinal effects of immunization or immunization plus chlorhexidine demonstrates that there is a clear shift in colonization. This approach uses a single immunization group study, and the participants in an Example 4 study may be used as a control. Although historical controls are often lirniting in value, the fact that the children may be enrolled from the same clinics should enhance the comparability of the results. Results are convincing that there is a substantial shift in the colonization before a randomized trial is undertaken. Thus, the basic approach analyzes saliva, oral rinse, and plaque samples to provide data on the presence of MS for correlations with immune responsiveness as well as colonization of permanent molar teeth following immunization with SBR-GLU, but may be modified as appropriate. An additional goal of this Example determines the effectiveness of mucosal immunization with SBR-GLU in inducing responses effective in protecting against dental caries of the newly erupted teeth.

The statistical hypotheses include:

The mean levels of salivary IgA against SBR, GLU, Agl/II, and GTF in the treated group up to three months following immunization are statistically significantly higher compared to the levels of same analyses in the historical control group;

The mean time to initial colonization of permanent molar teeth with mutans streptococci in the experimental group is statistically significantly longer compared to the time to initial colonization of mutans streptococci in the historical control group; The variability in colonization levels of newly erupted teeth is greater over the course of observation than established teeth;

The mean DMFS levels in the experimental group 5 years (only begin these analyses in this proposal due to timing and duration of implementation of this study, following the first children as long as possible to gain long term information useful for planning future studies) after immunization are statistically significantly lower compared to the mean DMFS in the control group; and

The incidence of adverse events in the experimental group are not a concern and do not differ significantly from the rates of occurrence found in Example 3.

The population for this study is assenting preadolescent and/or preschool aged children of consenting parents recruited, for example, from the School of Dentistry and Children's Hospital dental clinics (as recruited in Example 3 except the timing relative to molar eruption will be important also).

The sample size of 80 is chosen based on the following calculations: A sample size of 64 in each group (Treated Group and Historical Control) has 80% power to detect a difference in means of one half a standard deviation unit (the difference between the mean colonization time in the treated group should be half again longer than the mean time until colonization estimated from the control group from Aim 2) using a two group t-test with a 0.05 two-sided significance level. To be conservative, one may increase the sample size to 80 which is sufficient to detect a 45% increase in the time to colonization. Example 4 may indicate that a contemporaneous control group may not be efficient at this stage of investigation. Although 80 additional children necessary for this study, if a contemporaneous control is used, provides useful and interesting results on the natural history of colonization, it doubles the recruitment and logistics. This approach demonstrates large effects, lest more work on immune responses will be required. Optionally, a contemporaneous control group may be used.

Because of the small length of time involved in the proposed immunization (12 weeks) and previous experience with subject reliability, attrition is estimated to be less than 5% which will not cause problems in the proposed analyses. Further, the results from the studies in Example 4 provides better preliminary estimates of variation expected in this Example study, therefore, appropriate adjustments to sample size may be made based on these findings.

Variables of interest will be categorized into following areas: Demographic and ID variables: Name, DOB, gender, race, SSN (for follow-up and payment of incentives);

Immunogenicity related variables: Salivary IgA and serum IgA and IgG against SBR, GLU, Agl/II and GTF;

Intermediate end point variables: MS time to colonization, and MS levels. Molar eruption time(s) are used as a covariate in the analysis by recording it and adjusting for its effect. This facilitates more direct comparison of eruption time and time to colonization data. An incentive to cooperate with this request will be instituted to ensure compliance;

Timing of immunization and MS colonization of newly erupting molar teeth;

Interim end point related variable: time to colonization and caries prevalence; Adverse immune response: Serum IgG antibody activity against human heart sarcolemma and myosin preparation; and

Safety related variables: Fever, headache, malaise, rash, nasal congestion, rhinorrhea, stomatitis, and nausea, (as recorded on a diary by caregiver).

Note that if a new molar tooth does not erupt before the end of the period of time for active sample collection to monitor immune response, the parents and subject will be instructed on what to look for to document the beginning of eruption of permanent molar. They will be shown photographs of erupting teeth. Furthermore, the child will be encouraged to feel for the beginning of tooth eruption with their tongue. Additionally, the clinical coordinator will make monthly phone calls to the parent of each subject to determine if any new teeth have begun to erupt and/or remind the participants to be aware of any new tooth eruptions. When a new tooth is detected, they will be asked to document the first day and call for an appointment to come for sample collection. Because the time will be in addition to the routine sample collection, an extra incentive payment will be provided in addition to the normal reimbursement for sample collection.

A similar experimental approach is used as that presenting in Example 3. Regarding subject recruitment and specimen collection, children (age 5- 6 or 10-12 years), will be recruited to participate. Patients are enrolled from of clinics, such as those at a teaching hospital or university, as in Example 3. The selection criteria are also the same as in Example 3, but immunization will not begin until one permanent molar begins to erupt (and not more than two). The time to colonization, therefore, is the "next" tooth to erupt (i.e. after immunization). Regarding sample collection, parotid saliva and peripheral blood will be collected at intervals similar to those defined in Example 3. Initial oral rinse samples will be collected as before to obtain a baseline of MS levels. Plaque samples will be collected from each of four existing fully erupted molars (as in Example 4) and when new permanent molars begin to emerge (i.e., individual plaque samples will be collected). Regarding the immunization protocol, the route, antigen form and age group chosen from Example 3 is used. Antigen preparation is described hi Examples 1, 2, and 6. The appropriate dehydrated antigen dosage will be aliquoted into sterile tubes (or capsules) according to weight.

The following experimental proposal outlines the studies to be performed to investigate the effectiveness of the SBR-GLU antigen for induction of mucosal immune responses and the effectiveness of the immune response hi delaying colonization of erupting tooth surfaces.

Eighty 10-12 year old subjects are immunized by nasal spray with 120 μl L-SBR- GLU each side (62.5 μg), twice (7 day interval). As in Example 3, for seven days following immunization, subjects complete a daily diary with specific questions about any adverse events. If Example 4 results indicate the need to use an antimicrobial approach to lower the MS load, this protocol is instituted 2 weeks after immunization begins. Children are given a rubber-cup prophylaxis completed and then asked to rinse twice a day (after morning and night tooth brushing) with 0.12% chlorhexidine (Periogard^®, Colgate Pahnolive Inc., New York, NY) for 1 minute and expectorate without rinsing afterwards. Subjects are given 28 tubes clearly labeled for each rinse time. They are asked to return after 14 days for sample collection to determine the antimicrobial effectiveness and assessment of compliance of rinse schedule. An incentive may be given to insure compliance, e.g., $25 savings bond in the child's name, when the empty tubes are brought back for sample collection. Monthly microbial samples are then collected until newly erupted molar assessment is completed. Baseline specimens and analyses follow the schedule as described in Example 3.

Microbial samples are collected as in Example 4. After completion of the immunization regimen, parotid saliva and blood specimens are collected for assessment of immune response to MS as previously described in Example 3 bi-weekly for 5 weeks, then at monthly intervals for 2 additional months, followed by 3 month samples for 9 additional months.

Microbiological samples will be collected as described in Example 4 at each of the specimen collection time points as described in item c. above. Additionally, microbiological samples are collected monthly until newly erupted molar teeth are adequately assessed for colonization with MS (e.g., up to one year or more after initial immunization begins).

Laboratory personnel will analyze samples identified only by code which indicates sample type (i.e., saliva, serum, oral rinse, plaque) and date and subject number.

Bacteriological samples for enumeration of percent MS/total streptococci are handled as presented in Example 3, as is analysis for antibody activity by ELISA. Time for colonization of permanent teeth with MS and caries development in permanent molar teeth are also tracked. These steps are illustrated in the following summary table:

Time (relative to immunization, months)

Event 1 mo Day 0 l mo 2 mo 3 mo 6 mo 9 mo 12 18 24 30 36 before (and 7) (2)* mo mo mo mo mo

Eligibility X

Screening

Informed X

Consent

Dental Exam. X X

(DMFS)

Study X

Questionnaires

MS levels X X X X

(saliva)

Randomization X

Treatment/Plac X ebo

Saliva X X X X X X sample/IgA levels

Serum X X X X X X X sample/IgG levels

MS sample X X P P P P

(plaque)

Adverse events X X X X X X P P P P

* 2 samples collected biweekly during first month after immunization (i.e., days 21, 35) t "p^» indicates possible sampling depending on when recruited (e.g., limitation of 5 year period of the study)

The study is composed of subjects and an historical control (referred to for convenience as experimental and control) with repeated measures within subjects over time. Age, race, gender, and baseline MS percentage are potential covariates. The primary analysis consists of time until colonization between the treatment group and the historical data from Example 4. The time until colonization is longer (perhaps 50%) in the treatment group. There are many important within-group analyses for which there are repeated measures over time. Initially the primary interest is in the post-immunization mucosal immune response between the two groups. Within-group assessment of levels of change from the baseline period to delay colonization and the other covariates that impact these results (i.e. DMFS, MS levels and anti-MS levels before immunization, age, etc.) are also compared. In addition, the variability over time in colonization is assessed and compared for newly erupted teeth compared to established teeth. Analyses are conducted using generalized least squares analyses (PROC MIXED in SAS). Appropriate transformations of the data will be used to stabilize variance and to improve normality as needed. In some cases it may be useful to summarize the data in terms of a modeled curvilinear response function for each group with group comparisons among the coefficients of these curves. A type I error level of 5% will be used for the statistical tests.

Important data is obtained to determine the immunogenicity and effectiveness of the

5 immunization (altered MS colonization) of the experimental groups when given by a mucosal route to children. It is anticipated that a sufficient number of subjects complete the program to determine if a significant salivary immune response is induced. Additionally, it is important to monitor the effect of the immunization on colonization with MS as a surrogate for protection against dental caries. Furthermore, the prevalence of dental caries in the

0 population planned for study may not be sufficient for statistical power to show differences but will be monitored. This study determines the immune responsiveness of mucosal immunization to MS and furthermore, provide an indication of the timing of MS colonization when new molar teeth erupt and how salivary IgA anti-MS antibodies may modulate colonization.

[5

Example 6. Immunogenic responses elicited by SBR-GLU chimeric protein.

In addition to antigen-specific signals mediated through the T-cell receptor, T-cells also require antigen nonspecific costimulation for activation. The B7 family of molecules on antigen-presenting cells, which include B7-1 (CD80) and B7-2 (CD86), play important roles

20 in providing costimulatory signals for development of antigen-specific immune responses. See, e.g., Zhang et al., "Role of B7 Costimulatory Molecules in Immune Responses and T- Helper Cell Differentiation to Recombinant HagB from Porphyrinurias gingivalis " 72(2) Infection & Immunity 637-644 (2004).

Femurs from C57BL/6 wild type or TLR knockout mice (TLR2, TRL4 or other TLR) 5 knockout mice (8 to 12 weeks old) were collected by dissecting the rear limbs of each mouse, and the epiphyses were removed from each end of the femurs using scissors to expose the bone marrow. Using a 5 ml syringe with a 22-gauge needle, 15 ml of ice-cold Hank's balanced salt solution (HBSS) was used to aspirate the bone marrow out of the femur into a polystyrene Petri dish. The dendritic cells were generated from the bone marrow by culturing 0 the bone marrow cells in the presence of 20 ng/ml rGM-CSF (Atlanta Biologicals, Atlanta, GA) for 10 days in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L- glutamine, 50 μM 2-mercaptoethanol, 20 mM HEPES, 1 mM sodium pyruvate, 50 μg /ml penicillin, and 50 μg/ml streptomycin (RMPI 1640 complete medium) in a humidified 5% CO2 incubator at 37°C.

The resulting non-adherent dendritic cells were harvested after 6 days and the purity of the dendritic cell population was determined by FACS analysis of CDl lc+ cells using a FACScaliber (Immunocytometry Systems, San Jose, Calif). This procedure routinely results in > 70% of the cells staining positive for CDl lc+. The dendritic cells (2 x 105 cells/culture) were incubated with various concentrations of SBR-GLU or control antigen in RPMI 1640 complete medium, in a humidified CO2 incubator at 37°C for 24 to 48 hours. Following incubation, the cells were harvested and stained with CDl Ic allophycocyanin (APC), counter-stained with phycoertherin (PE)-conjugated anti-mouse B7-1 and fluorescein isothiocynate (FITC)-conjugated anti-mouse B7-2 (eBioscience, San Diego, Calif), and analyzed for B7-1 and B7-2 expression by FACS.

The levels of IL-10, IL-12p40, IL-12p70, IFN-γ and TNF-α in the culture supernatants were determined by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (IL-10, IL-12p40 and IL-12p70 ELISA kits, BD Biosciences; IFN- γ and TNF- α ELISA kits, eBioscience, San Diego, CA).

The results indicate that the SBR-GLU chimeric protein signals through the TLR 4 pathway. Exposure of dendritic cells to the SBR-GLU chimeric protein resulted in an up- regulation of B7-1, B7-2, and CD40, as well as the induction of TNF-α, IL-10, and IL-12p40 production.

Claims

We claim:

1. A vaccine that generates a protective response to mutans streptococci in a mammalian host, wherein said vaccine comprises a therapeutically effective amount of SBR-GLU, or a chimeric polypeptide containing immunogenic portions of each of said SBR and GLU₅ wherein said SBR-GLU comprises the immunogenic regions of the mutans streptococci virulence factors Agl/II and glucosyltransferase.

2. The vaccine of claim 1, wherein said mutans streptococci is a Streptococcus mutans isolate or a S. sobrinus isolate.

3. The vaccine of claim 1, wherein said vaccine is delivered to the mucosae.

4. The vaccine of claim 1, wherein said vaccine stimulates a mucosal immune response.

5. An immunogenic polypeptide comprising chimeric SBR-GLU, or a chimeric polypeptide containing immunogenic portions of each of said SBR and GLU, wherein said SBR-GLU comprises the immunogenic regions of the mutans streptococci virulence factors, Agl/II and glucosyltransferase.

6. An immunogenic composition that generates an immune response to mutans streptococci in a mammalian host, comprising the immunogenic polypeptide defined in claim 5 and a physiologically acceptable carrier.

7. The immunogenic composition defined in claim 6 further comprising an adjuvant.

8. An immunodiagnostic for the detection of mutans streptococci comprising the immunogenic polypeptide defined in claim 5.

9. An immunodiagnostic kit for the detection of mutans streptococci in a test subject comprising a) the immunogenic polypeptide defined in claim 5; b) a suitable support phase coated with SBR-GLU; and c) labeled antibodies immunoreactive to antibodies from said test subject.

10. An isolated DNA polynucleotide encoding a chimeric polypeptide SBR-GLU or a chimeric polypeptide containing immunogenic portions of each of said SBR and GLU, wherein said SBR-GLU comprises the immunogenic regions of the mutans streptococci virulence factors, AgI/II and glucosyltransferase.

11. A vector comprising the DNA polynucleotide of claim 10.