Streptococcus mutans Infection after Intranasal - Infection and ...

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Dec 21, 1992 ... We thank Noel K. Childers, John Eldridge, and Dawn C. Ward for their .... CZ. (U cn co. C* u. 0 r. 0 cns. 0 co. U. C. (U. *(U. U. *. 'e. 0. -o. C. (U. C.

Vol. 61, No. 5

INFECTION AND IMMUNITY, May 1993, p. 1964-1971

0019-9567/93/051964-08$02.00/0 Copyright © 1993, American Society for Microbiology

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 JANNET KATZ, CECILY C. HARMON, GERRA P. BUCKNER, GLORIA J. RICHARDSON, MICHAEL W. RUSSELL, AND SUZANNE M. MICHALEK* Departments of Microbiology and Oral Biology, The University of Alabama at Birningham, Binningham, Alabama 35294

The B subunit of cholera toxin (CTB) has been shown to augment mucosal responses to microbial virulence antigens, including those of Streptococcus mutans, which is the principal etiologic agent of dental caries. In the present study, the surface fibrillar protein antigen of S. mutans, antigen I/II (Ag I/I), was chemically coupled to CTB (Ag I/I-CTB), and the conjugate was examined for its effectiveness in inducing salivary immune responses protective against S. mutans infection. Weanling Fischer rats were given Ag I/II-CTB (50 p,g) by the intranasal route and then orally infected with a virulent strain of S. mutans. Gnotobiotic or conventional rats were given two or three additional immunizations, respectively, at about 2-week intervals. One week after each immunization, individual serum, saliva, and fecal samples were collected and stored frozen until assayed for antibody activity to Ag IJI and cholera toxin (CT) by an enzyme-linked immunosorbent assay. The rats were sacrificed 1 week after the last immunization, when mandibles were also collected from individual rats for assessment of S. mutans levels in plaque and caries activity. Rats immunized only or both immunized and infected showed a salivary immunoglobulin A (IgA) anti-Ag I/Il response which reached significantly (P < 0.05) higher levels than those seen in nonimmunized, infected controls. A salivary IgA anti-Ag I/II response was also seen in rats infected only with S. mutans. Essentially no salivary antibody activity to CT was detected. Some serum anti-Ag VII and anti-CT responses were seen in immunized animals. Serum IgG anti-Ag IJI responses were seen in immunized, infected rats and also in infected-only rats, suggesting that the responses were a result of infection with S. mutans. The immunized and infected rats had significantly (P < 0.05) lower levels of S. mutans in plaque and lower caries activity than nonimmunized, infected rats. These results indicated that intranasal immunization of rats with Ag I/I-CTB induced a protective salivary immune response which was associated with a reduction in S. mutans colonization and S. mutans-induced dental caries.

the mucosal immune responses after intranasal (i.n.) immunization. Studies involving the oral or nasal methods of antigen delivery have used cholera toxin (CT) or its nontoxic pentameric B subunit (CTB) as an immunogen and/or adjuvant (4, 7, 35, 36, 40). The adjuvant property is partly explained by the ability of CTB to bind to GM, ganglioside, a membrane component present on mammalian cells (3, 16). Therefore, by genetic or chemical coupling to CTB, the mucosal immunogenicity of microbial antigens can be enhanced without the noxious effects caused by the toxic A subunit of CT (4, 5, 32). In the study reported here, we have used the microbial surface protein antigen I/II (Ag I/II) chemically coupled to CTIB (Ag I/II-CTB) for i.n. immunization. Ag I/II (also known as antigen B, P1, and PAc [1, 27, 34]) is a surface fibrillar protein of the cariogenic organism Streptococcus mutans (28) and is a virulence factor that contributes to the pathogenesis of S. mutans-induced dental caries because of its involvement in the initial adherence of the organism to tooth surfaces and in aggregation (11). Evidence has been presented that oral administration of Ag I/II to rats results in the appearance of secretory IgA antibodies in saliva and a reduction in S. mutans colonization and protection against caries formation (23). Other investigations in mice have shown that intragastric administration of the Ag I/II-CTB

The common mucosal immune system includes distinct immunoglobulin A (IgA)-inductive and -effector sites (2, 22). In the gut, the IgA-inductive sites are referred to as the gut-associated lymphoid tissue and include the Peyer's patches (22). The IgA-inductive tissues in the respiratory tract are distributed in the bronchi (the bronchial-associated lymphoid tissue) and the so-called Waldeyer's ring, which consists of the tonsillar structures in humans (20). In rodents, the tissue present in the floor of the nasal cavity on both sides of the entrance to the nasopharyngeal duct, considered to be equivalent to the Waldeyer's ring in humans, is known as the nasal-associated lymphoid tissue (19, 20). In humans and other mammals, the predominant immunoglobulin in external secretions is secretory IgA, which is a first line of defense of mucosal surfaces against microbial pathogens (22, 31). This finding has led to numerous investigations to determine the safety and effectiveness of various procedures for the induction of specific secretory IgA antibodies in external secretions. Among the methods tested, the induction of a mucosal immune response by intragastric administration of antigen has been most extensively documented (22, 31). However, less is known about the nature of


Corresponding author. 1964

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Received 21 December 1992/Accepted 18 February 1993

VOL. 61, 1993


conjugate induces a higher salivary (secretory IgA) response than that seen in animals given antigen alone (4, 32). Furthermore, i.n. administration of the conjugate to mice results in higher salivary responses to Ag I/II than that seen in mice immunized by the oral route (41). The purpose of the present study was to determine if i.n. immunization of conventional and gnotobiotic rats with Ag I/II-CTB would induce a salivary IgA antibody response and protection against S. mutans colonization and caries formation. (This work was presented in part at the Seventh International Congress of Mucosal Immunology, Prague, Czechoslovakia, August 1992.)

Systems, Cockeysville, Md.). Antigens. S. mutans Ag I/II was isolated and purified from IB162 and conjugated to CTB as described previously (29, 32). Briefly, equimolar amounts of Ag I/II and CTB (List Biological Laboratories, Inc., Campbell, Calif.) were coupled by using N-succinimidyl-(3-[2-pyridyl]-dithio) propionate (Pharmacia LKB Biotechnology, Piscataway, N.J.). The conjugate was dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at -70°C. Enzyme-linked immunosorbent assay (ELISA) of plates coated with GM, ganglioside showed that both receptor binding activity of CTB and antigenicity of Ag I/II were preserved in the conjugates. The Ag I/II-CTB molar ratio was approximately 1:1. CT was purchased from List Biological Laboratories. Bovine GM, ganglioside was obtained from CalbiochemBehring (San Diego, Calif.). Experimental rat model. Germ-free (termed gnotobiotic after infection with a specific organism) Fischer rats [CD F(344)GN/Crl BR] used in these studies were bred and maintained in Trexler plastic isolators at the University of Alabama at Birmingham Gnotobiotic Rat Facility (25, 26). Conventional weanling Fischer rats were purchased from Charles River Laboratories (Wilmington, Mass.). All experimental gnotobiotic and conventional rats were maintained in covered sterile cages in a laminar-flow hood throughout the experiment and provided cariogenic diet 305 and water ad libitum (25). All animal experiments performed in these studies were approved by the University of Alabama at Birmingham Animal Resources Advisory Committee. Experimental design. Weanling Fischer rats (age, 19 to 20 days; six per group) were given Ag I/II-CTB (50 ,ug of total protein in 50 p,l of PBS per rat) by the i.n. route on day 0. The conjugate was administered by introducing 12.5 ,u1 into each nostril with the aid of a pipettor (100 p,l) adapted with a sterile tip. This procedure was repeated once, so that each nostril received 25 ,ug of protein. Rats were then challenged (by oral swabbing) with S. mutans UA130 (on days 3 and 4) after the initial immunization (group B). Gnotobiotic rats were given additional i.n. immunizations of Ag I/II-CTB (50 p.g) on days 14 and 30, while conventional rats were given additional i.n. immunizations of Ag I/II-CTB (50 p.g) on days

14, 34, and 48. Control groups of rats (five per group) consisted of immunized and noninfected (group A), nonimmunized and infected (group C), and nonimmunized and noninfected (group D) animals. Serum, saliva, and fecal samples were collected from individual animals 7 days after each immunization and assayed for antibody activity to Ag I/II and CT by ELISA (see below). Mandibles were aseptically removed from rats at the termination of the experiment (day 37 for gnotobiotic rats and day 55 for conventional rats) and assessed for the number of S. mutans organisms present and for the level of caries activity on molar surfaces (25, 26). Briefly, the right mandible from each rat was transferred to tubes containing 3 ml of sterile phosphate buffer (0.067 M, pH 7.2). The plaque was disrupted from the molar surfaces by sonication (Branson Instruments Co., Plainview, N.Y.), and the numbers of S. mutans and total bacteria in plaque were determined after culturing serial 10-fold dilutions of samples on mitis salivarius and blood agar, respectively. The right and left mandibles were then cleaned, stained with murexide (0.4% in 70% ethanol), and hemisectioned, and buccal, sulcal, and proximal molar caries were scored by the procedure of Keyes


Collection of fluids. Blood was collected from the retroorbital plexus during the experimental period and by cardiac puncture at the termination of the experiment while rats were under anesthesia (100 mg of ketamine plus 1.5 mg of xylazine per ml; 0.1 ml/100 g of body weight via the intraperitoneal route). The blood was allowed to clot at 4°C, and after centrifugation, the serum was collected and stored at -20°C until assayed for antibody activity by ELISA (described below). Saliva was collected with the aid of a Pasteur pipette after injection of carbachol (Sigma Chemical Co., St. Louis, Mo.; 5 jig in 0.05 ml, intraperitoneally) to stimulate flow. Saliva was clarified by centrifugation and stored at -20°C until assayed for antibody activity. Fresh fecal pellets (three to four) were collected from each animal in separate tubes and processed as described previously (6). Briefly, to each tube was added 1 to 2 ml of PBS containing 0.01% sodium azide. The tubes were vortexed, and after 15 min of settling time the tubes were vortexed again. After this procedure was repeated once more, the samples were centrifuged (13,000 x g, 10 min, 4°C) and the supernatants were collected and stored at -70°C until assayed for antibody


ELISA. An ELISA was used to assess antibody activity to

Ag I/II and CT as described previously (17, 26, 32). Briefly,

individual wells of flat-bottom 96-well plates were coated with Ag I/II (5 p,g/ml) or with GM, ganglioside (2.5 p,g/ml) and then CT (1 ,ug/ml). Four twofold dilutions of serum, saliva, or fecal samples or six twofold dilutions of a serum or saliva pool standard were added in duplicate to individual wells, and the plates were incubated for 2 h at room temperature. Biotin-conjugated anti-rat immunoglobulin,

IgM, IgG (Southern Biotechnology Associates, Birmingham, Ala.), or IgA (Zymed Laboratories, Inc., San Francisco, Calif.) was added to appropriate wells, and then streptavidin-alkaline phosphatase (0.4 mg/ml; Southern Biotechnology Associates) was added. After the addition of the phosphatase substrate (Sigma 104) in diethanolamine buffer, color development was recorded at 405 nm in a Vmax microplate reader (Molecular Devices Corp., Menlo Park, Calif.) interfaced with a Macintosh SE computer. A standard curve of antibody activity (in ELISA units [EU]) was established by using a serum or saliva pool standard which was assigned a level of activity (in EU per milliliter),

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MATERIALS AND METHODS Bacteria. S. mutans IB162 (obtained from Kenneth W. Knox, United Dental Hospital, Sydney, Australia), which secretes Ag I/II (29, 32), and UA130 (obtained from Howard K. Kuramitsu, University of Texas Health Center, San Antonio), which has been shown to be virulent in our experimental rat model (14), were stored in glycerol-brain heart infusion (glycerol-BHI) broth (Difco Laboratories, Detroit, Mich.) at -70°C until used. S. mutans UA130 cultures used for infection were grown in BHI broth for 18 h at 37°C in an anaerobic GasPak System (BBL Microbiology




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