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Humans Immunized with Streptococcus mutans Antigens by Mucosal Routes
1 Department of Pediatric Dentistry and Correspondence: *corresponding author, nkc{at}uab.edu
Strategies aimed at the prevention of Streptococcus mutans infection and dental caries include mucosal immunization, which results in salivary anti-S. mutans responses. The purpose of this study was to evaluate the effectiveness of nasal vs. tonsillar immunization with S. mutans antigens in inducing salivary immune responses. Twenty-one adult subjects were immunized twice, within a seven-day interval, with a glucosyltransferase-enriched preparation (E-GTF) administered by nasal or tonsillar topical spray. Parotid saliva, nasal wash, and serum were collected prior to and at one- to two-week intervals for 3 months following immunization and were assayed by ELISA for anti-E-GTF activity. Results were analyzed by means of the mixed-models procedure with p < 0.05 level of significance. Significantly higher anti-E-GTF responses were detected in saliva and nasal wash samples from the group immunized by the nasal compared with the tonsillar route, indicating that nasal immunization was more effective in inducing mucosal responses in adults.
Key Words: mucosal immunity • immunization • caries • liposomes • Streptococcus mutans
Since the demonstration that dental caries is an infectious disease in which Streptococcus mutans has been implicated as a principal etiologic agent, interest has focused on the development of a vaccine that would prevent caries (Russell et al., 1999). Previous human studies designed to induce salivary IgA immune responses to S. mutans antigens have used the oral (Childers et al., 1991, 1994; Smith and Taubman, 1987,1990) and nasal (Childers et al., 1997,1999) routes for immunizations. Although results have been encouraging, salivary responses were variable, transient, and low in magnitude. Therefore, we have begun to investigate the effectiveness of other mucosal routes for administration of S. mutans antigen vaccines to induce salivary immune responses in FDA Phase I clinical trials. Evidence has been presented that tonsils may play a role in the selective induction of oral responses (Fukuizumi et al., 1995). Studies in rabbits found that tonsillar immunization with S. mutans resulted in the induction of a salivary response (Fukuizumi et al., 1997). In humans, immune responses following intra-tonsillar immunization were compared with intranasal, oral, and parenteral immunization (Quiding-Jarbrink et al., 1995). Subjects immunized by the tonsillar route had a higher number of specific antibody-secreting cells in removed tonsils than seen in subjects immunized by the other routes. These studies and others indicate that IgA-inductive sites (i.e., tonsils) of the common mucosal immune system (CMIS) may preferentially supply IgA-committed, antigen-sensitized cells to local mucosal regions (Brandtzaeg, 1984; Fukuizumi et al., 1995; Moldoveanu et al., 1995; Quiding-Jarbrink et al., 1995). The purpose of this study was to determine the effectiveness of a topical application of an S. mutans vaccine to the tonsils vs. nasal surfaces of adult volunteers in inducing mucosal immune responses. In this study, we used soluble and liposomal S. mutans antigens to immunize humans in a double-blind study.
Bacteria, Media, Immunogen Preparation We used S. mutans strain GS-5 (a serotype c isolate, obtained from F. Macrina, Virginia Commonwealth University, Richmond, VA, USA) to derive the E-GTF as previously described (Childers et al., 1997) from a large batch of GS-5 grown in 400 liters of broth culture media (CDM media, J.R.H. Biosciences, Lenexa, KS, USA) at the UAB Fermentation Facility. This material was used as the soluble antigen preparation in this and other Phase I human studies (Childers et al., 1994, 1997, 1999). The main components of E-GTF have been shown to be GTF and truncated AgI/II (Childers et al., 1999).
We prepared the liposomal vaccine by sonicating the appropriate amount of E-GTF in a flask coated with a lipid monolayer consisting of D,L-
Study Design
Unstimulated parotid saliva, nasal wash, and serum samples were collected weekly for 3 wks prior to immunization (baseline). Each subject was immunized with 125 µg of E-GTF delivered in a total volume of 244 µL to each site (Fig. 1
Sample Collection Parotid saliva samples were obtained by means of Schaefer cups (Schaefer et al., 1977). We obtained nasal wash samples by depositing 1.5 mL of sterile saline into each nostril of a reclining subject. After approximately 10 sec, the subject was instructed to sit up, and the nasal wash solution was allowed to drain into a specimen cup. Saliva and nasal wash samples were clarified by centrifugation at 14,0003g in an Eppendorf centrifuge. Serum was obtained after centrifugation (14,0003g) of blood collected by finger stick into a microvette tube with clotting activator (Sarstedt, Numbrecht, Germany). All samples were aliquoted and frozen at -70°C until used for ELISA.
Antibody Analysis
Statistics
All 21 individuals were followed for at least 3 mos following immunization. Other than transient headache (five subjects), mild nausea (five subjects), and nasal congestion (five subjects) that were not definitely attributed to the study, none of the subjects reported any side-effects during or after immunization (13 subjects reported no side-effects). Additionally, 12 subjects (57%) returned 18 mos following immunization for sample collection.
Nasal Anti-E-GTF Responses
The range of nasal IgA anti-E-GTF antibody activity in all subjects was 13.4-887.7 ng/mL, and the total IgA range was 5.7-246.9 µg/mL. In mixed-model analysis, there were significant differences between the two sites (IN > IT, p < 0.05). A nasal wash IgA anti-E-GTF response was seen in the IN but not the IT immunized group (Fig. 2  , bottom). The response in the IN group peaked on day 21 (154% increase over baseline) following immunization. The predominant subclass of the observed response in nasal wash was IgA1. The IgA anti-E-GTF response in nasal secretion persisted for up to 18 mos post-immunization in the IN group (data not shown). Furthermore, the IgA1 anti-E-GTF/total IgA response in IN subjects at 18 mos was significantly higher (t test, p < 0.0138) than that seen in the IT subjects.
Saliva Anti-E-GTF Responses
Serum Anti-E-GTF Responses A serum IgA response was detected in eight of 11 IN and three of ten IT subjects, with an increase of at least 100% over baseline. The IgA anti-E-GTF response was detected on day 28 in the IN group and persisted through day 90 (mean increase 100 to 200% over baseline; Fig. 4  ). Due to individual variation, the mean response values were not significantly different from those obtained in pre-immune samples, nor was the IgA anti-E-GTF response different between soluble vs. L-E-GTF and IN vs. IT groups. Little or no increase over baseline IgA anti-E-GTF activity was seen in the 18-month samples (data not shown).
The mean serum IgG response was higher (~ 40% mean increase over baseline; Fig. 4 ) in the IN group but not significantly different from that seen in the IT group. Only four of 21 subjects showed an increase of more than two-fold over baseline anti-E-GTF activity. The responses were not significantly increased from baseline, nor were the 18-month samples significantly different from baseline (data not shown).
We have recently reported the effectiveness of nasal immunization of humans with S. mutans antigens in inducing nasal and salivary responses (Childers et al., 1999). The purpose of this study was to compare responses induced following the topical application of S. mutans vaccine to tonsils with those seen following nasal immunization. This was a double-blind clinical study testing two forms of the vaccine antigen, i.e., soluble and liposomal antigens, and used a dose of E-GTF which was smaller than that previously used for nasal immunization (Childers et al., 1999). The results of this study and previous studies (Childers et al., 1997, 1999) provide evidence for the safety of both soluble and liposomal E-GTF for use in humans, and that these vaccines were immunogenic when given by the IN route. A significantly higher mean post-immunization IgA anti-E-GTF response was observed in nasal wash and parotid saliva but not in serum of the IN groups compared with the IT immunization groups. In the present study, the subjects given L-E-GTF by the IN route had higher IgA responses compared with responses seen in individuals given soluble E-GTF; however, the differences were not significant. We have previously shown that subjects given liposomal E-GTF by the IN route had significantly higher nasal wash IgA1 immune responses than those seen in subjects given soluble antigens (Childers et al., 1999). It is possible that the lack of statistical significance in the responses observed between the two groups was due to the lower number of individuals in each group (five vs. ten) and the lower dose of E-GTF (i.e., 125 µg vs. 250 µg) than used in our previous study. Our previous studies have followed immune responses for only 3 mos. In this study, we have shown that nasal wash responses persisted for up to 18 mos in subjects immunized via the nasal route. This finding is important from a practical aspect of vaccine design, to minimize the number of booster immunizations needed for protection. A long-term goal of our studies is to identify a mucosal route of immunization with an S. mutans vaccine to induce effective, persistent salivary immune responses. Although a significant increase was seen in the salivary IgA response after IN immunization, the response was lower than that seen in nasal wash. This finding provides support for compartmentalization within the CMIS, which results in differential responses at different secretory sites (Brandtzaeg, 1984; Moldoveanu et al., 1995; Quiding-Jarbrink et al., 1995; Fukuizumi et al., 1999, 2000). Nasal immunization was a more effective immunization route than IT in the studies reported herein. These findings were in contrast to the findings of Fukuizumi and co-workers (Fukuizumi et al., 1995), who evaluated responses in rabbits that were immunized by the nasal vs. tonsil routes and found that IN immunization resulted in nasal responses, while IT immunization resulted in salivary responses. Human tonsillar immunization studies have evaluated the effect of antigen injection (Quiding-Jarbrink et al., 1995) rather than topical antigen application as evaluated here. Although it may be that topical application of antigen to tonsils is not a useful method for immunization in adults, it cannot be ruled out for use in children. Children may have a better potential for responding to a tonsillar immunization, since adults experience senescence of tonsil tissue function. Therefore, this type of tonsil immunization study is needed in children so that the potential for topical immunization can be determined. In this study, nasal and tonsillar immunization was found to be safe in humans. Furthermore, IN immunization of humans with soluble or liposomal E-GTF antigens from S. mutans resulted in immune responses in nasal secretions and parotid saliva. Additional studies are needed to identify ways of inducing predominantly salivary IgA responses and to determine the ability of induced IgA responses to modulate dental colonization with S. mutans so that an effective approach to the prevention of dental caries can be designed. Since a dental caries vaccine may be most effective in younger populations, now that safety data have been accumulated for adults, studies in younger populations are needed. In this regard, studies in children are needed to identify a mucosal immunization route, dosage, antigen form, adjuvant, and timing schedule for optimal enhancement of the magnitude and longevity of the salivary responses that have been observed in adults.
The authors thank Dr. Jiri Mestecky for providing colostral IgA and Pfeiffer of America for providing the Bi-Dose System spray devices. We also thank Ms. Rosie Turner for secretarial help. This work was supported in part by USPHS Research Grants DE09846, DE09081, and DE08182 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, 20892. Received for publication June 26, 2001. Revision received November 9, 2001. Accepted for publication November 14, 2001.
Journal of Dental Research, Vol. 81, No. 1,
48-52 (2002) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
-dipalmitoyl phosphatidylcholine, cholesterol, and dicetylphosphate (Sigma Chemical Company, St. Louis, MO, USA) and then filtering through a 100-nm-pore membrane (LiposoFast, Avestin, Inc., Ottawa, ON, Canada), as previously described (
indicates days of immunization.
0.05 was considered as the accepted level of significance. 
-, n = 6) or L-E-GTF plus standard deviation (Group BN; -
-, n = 5) on days 0 and 7. IgA anti-E-GTF antibody activity in nasal wash secretions from IN (-•-) and IT (-
-) immunized subjects (bottom panel). Values are the mean ratio of IgA E-GTF/total IgA (plus standard deviation) for samples collected before and after immunization with 125 µg S. mutans antigens (combined data from soluble E-GTF and L-E-GTF groups) on days 0 and 7. Mixed-model analysis resulted in a significant difference between responses in IN (n = 11) vs. IT (n = 10) groups (p < 0.05, see RESULTS).
