HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (17) Citing Articles via Google Scholar Google Scholar Articles by Childers, N.K. Articles by Michalek, S.M. Search for Related Content PubMed PubMed Citation Articles by Childers, N.K. Articles by Michalek, S.M.

Humans Immunized with Streptococcus mutans Antigens by Mucosal Routes

¹ Department of Pediatric Dentistry and
² Oral Biology, School of Dentistry, Room 308, 1530 3rd Ave. South, University of Alabama at Birmingham, Birmingham, AL, USA 35294-0007;
³ Department of Biostatistics, School of Public Health, University of Alabama at Birmingham; and
⁴ Department of Microbiology, University of Alabama at Birmingham;

*corresponding author, nkc{at}uab.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
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

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
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.

	MATERIALS & METHODS

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
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--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 (Childers et al., 1997).

Study Design
Twenty-one healthy adult volunteers from 20 to 50 yrs of age were recruited. In compliance with guidelines established by the UAB Institutional Review Board, written informed consent was obtained from each subject. All subjects had previous experience with dental caries, although none had active caries lesions prior to or during the study. The subjects were randomly assigned to one of four groups balanced for age and sex.

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). The intranasally (IN) immunized subjects received 122 µL (62.5 µg) of vaccine deposited into each nostril by means of a Bi-Dose System nasal spray (Pfeiffer, Princeton, NJ, USA), while the subject was in a reclined position. Similarly, for tonsillar (IT) immunization, the vaccine was sprayed onto each palatine tonsillar pillar. Following immunization, samples were collected weekly for 6 wks and on days 56 and 90, and at 18 mos (day 540). All samples were analyzed by ELISA for anti-E-GTF antibody. Three additional pre-immunization samples were available for 16 of the 21 subjects. These samples were obtained during an initial screening one year prior to the beginning of the study.

View larger version (5K): [in this window] [in a new window]   Figure 1. Study design. The subjects were immunized via the IN or IT route with either soluble E-GTF or liposomal E-GTF (L-E-GTF). Subjects were randomly assigned to group AN (soluble E-GTF, IN, n = 6), AT (soluble E-GTF, IT, n = 5), BN (L-E-GTF, IN, n = 5), or BT (L-E-GTF, IT, n = 5). Samples of parotid saliva, nasal wash, and blood were collected on days indicated by vertical lines. indicates days of immunization.

Antibody Analysis
We used an ELISA to determine the levels of total immunoglobulin and the relative concentrations of antibodies to S. mutans E-GTF as previously described (Childers et al., 1994). Optimal dilutions of saliva, nasal wash, or serum in duplicates of 4 to 8 two-fold dilutions were added to designated wells of E-GTF (2.5 µg/mL)-coated microtiter plates. A human serum pool of known isotype concentrations (Dade Moni-trol, Baxter Diagnostic Inc., Deerfield, IL, USA) and purified human colostral IgA (provided by J. Mestecky, University of Alabama at Birmingham) were used as the immunoglobulin standards. A four-parameter curve-fitting program (Softmax^TM, Molecular Devices, Menlo Park, CA, USA) was used to construct reference curves for each ELISA plate from O.D. readings of the known immunoglobulin standard (i.e., Moni-trol or colostral IgA for serum or secretions, respectively). Serum results were reported as ng/mL anti-E-GTF antibody activity, while saliva and nasal wash results were converted to a ratio of anti-E-GTF per total IgA to normalize for variation in total immunoglobulin content in the samples. Results obtained from the 3 or 6 baseline samples were averaged for comparison with post-immunization anti-E-GTF responses (reported as percent increase over baseline activity for each subject).

Statistics
A mixed-model analysis was used for comparison of antibody activity. This analysis considers group (soluble vs. liposomal antigens), IN vs. IT route, time (i.e., pre-immunization vs. post-immunization), and ‘group X time' to be fixed effects, and subject-to-subject variation and its interactions to be random effects. Antibody levels were log-transformed to normalize for variance; however, graphic results are presented non-transformed. Two-sided type I error probability 0.05 was considered as the accepted level of significance.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
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
Subjects immunized with soluble E-GTF (Group A) vs. L-E-GTF (Group B) showed no difference in responses, except within the nasally immunized groups, where individuals given L-E-GTF had higher, but not significantly different, nasal IgA responses than those given soluble E-GTF (Fig. 2, top panel). Since no significant difference was seen between groups given soluble or L-E-GTF, the data from these groups were combined for further analysis.

View larger version (47K): [in this window] [in a new window]   Figure 2. IgA anti-E-GTF antibody activity in nasal wash secretions from IN immunized subjects (top panel). Values are the mean ratio of IgA E-GTF/total IgA for samples collected before and after immunization with 125 µg of soluble E-GTF minus standard deviation (Group AN; --, 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).

Saliva Anti-E-GTF Responses
The range of parotid saliva IgA anti-E-GTF antibody activity for all the subjects was 22.2-8806 ng/mL, and the total IgA range was 16.9-3405 µg/mL. No differences were found between groups given soluble (A) or L-E-GTF (B) (Fig. 3, top); therefore, data were combined for all IN and all IT immunized subjects. A mean peak of 71% increase in IgA anti-E-GTF activity over baseline occurred on day 35 (Fig. 3, bottom) in the IN group compared with 15% on day 21 in the IT group. The differences observed between the IN and IT groups following immunization were significant (p < 0.05). Little or no anti-E-GTF response was apparent 18 mos following immunization in parotid saliva (data not shown). Subclass analysis indicated a predominantly IgA1 subclass response in the IN group (data not shown).

View larger version (47K): [in this window] [in a new window]   Figure 3. IgA anti-E-GTF antibody activity in parotid saliva from IN immunized subjects (top panel). Values are the mean ratio of IgA E-GTF/total IgA for samples collected before and after immunization with 125 µg of soluble E-GTF minus standard deviation (Group AN; --, 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 parotid saliva from IN (-•-) and IT (--) immunized subjects (bottom panel). Values are the mean ratio of E-GTF-specific/total IgA anti-E-GTF activity in parotid saliva samples collected from IN (plus standard deviation) and IT (minus standard deviation) immunized subjects (combined data from soluble E-GTF and L-E-GTF groups). Mixed-model analysis resulted in a significant difference between responses in IN (n = 11) vs. IT (n = 10) groups (p < 0.05, see RESULTS).

View larger version (42K): [in this window] [in a new window]   Figure 4. IgG (top panel) and IgA (bottom panel) anti-E-GTF antibody activity in serum from IN (-•-) or IT (--) immunized subjects. Values are the mean levels (ng/mL) of anti-E-GTF activity before and after immunization (plus or minus standard deviation for IN or IT immunization, respectively). Data are combined from soluble E-GTF and L-E-GTF groups. Mixed-model analysis resulted in no significant difference between responses in IN (n = 11) vs. IT (n = 10) groups for IgG and IgA (p > 0.05, see RESULTS).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
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.

	ACKNOWLEDGMENTS

 
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 June 26, 2001; Last revision November 9, 2001; Accepted November 14, 2001

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Brandtzaeg P (1984). Immune functions of human nasal mucosa and tonsils in health and disease. In: Immunology of the lung and upper respiratory tract. Bienenstock J, editor. New York, NY: McGraw-Hill Book Co., pp. 28-95.

Childers NK, Michalek SM, Pritchard DG, McGhee JR (1991). Mucosal and systemic responses to an oral liposome-Streptococcus mutans carbohydrate vaccine in humans. Reg Immunol 3:289–296.

Childers NK, Zhang SS, Michalek SM (1994). Oral immunization of humans with dehydrated liposomes containing Streptococcus mutans glucosyltransferase induces salivary immunoglobulin A2 antibody responses. Oral Microbiol Immunol 9:146–153.[Medline]

Childers NK, Tong G, Michalek SM (1997). Nasal immunization of humans with dehydrated liposomes containing Streptococcus mutans antigen. Oral Microbiol Immunol 12:329–335.[Medline]

Childers NK, Tong G, Mitchell S, Kirk K, Russell MW, Michalek SM (1999). 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. Infect Immun 67:618–623.[Abstract/Free Full Text]

Fukuizumi T, Inoue H, Anzai Y, Tsujisawa T, Uchiyama C (1995). Sheep red blood cell instillation at palatine tonsil effectively induces specific IgA class antibody in saliva in rabbits. Microbiol Immunol 39:351–359.[Medline]

Fukuizumi T, Inoue H, Tsujisawa T, Uchiyama C (1997). Tonsillar application of killed Streptococcus mutans induces specific antibodies in rabbit saliva and blood plasma without inducing a cross-reacting antibody to human cardiac muscle. Infect Immun 65:4558–4563.[Abstract]

Fukuizumi T, Inoue H, Tsujisawa T, Uchiyama C (1999). Tonsillar application of formalin-killed cells of Streptococcus sobrinus reduces experimental dental caries in rabbits. Infect Immun 67:426–428.[Abstract/Free Full Text]

Fukuizumi T, Inoue H, Tsujisawa T, Uchiyama C (2000). Streptococcus sobrinus antigens that react to salivary antibodies induced by tonsillar application of formalin-killed S. sobrinus in rabbits. Infect Immun 68:725–731.[Abstract/Free Full Text]

Moldoveanu Z, Russell MW, Wu HY, Huang W-Q, Compans RW, Mestecky J (1995). Compartmentalization within the common mucosal immune system. In: Advances in mucosal immunology. Mestecky J, Jackson S, Kiyono H, McGhee JR, Michalek SM, Russell MW, et al., editors. New York, NY: Plenum Press Publishing Corporation, pp. 97-101.

Quiding-Järbrink M, Granström G, Nordström I, Holmgren J, Czerkinsky C (1995). Induction of compartmentalized B-cell responses in human tonsils. Infect Immun 63:853–857.[Abstract]

Russell MW, Hajishengallis G, Childers NK, Michalek SM (1999). Secretory immunity in defense against cariogenic mutans streptococci. Caries Res 33:4–15.[Medline]

Schaefer ME, Rhodes M, Prince S, Michalek SM, McGhee JR (1977). A plastic intraoral device for the collection of human parotid saliva. J Dent Res 56:728–733.[Abstract/Free Full Text]

Smith DJ, Taubman MA (1987). Oral immunization of humans with Streptococcus sobrinus glucosyltransferase. Infect Immun 55:2562–2569.[Abstract/Free Full Text]

Smith DJ, Taubman MA (1990). Effect of local deposition of antigen on salivary immune responses and reaccumulation of mutans streptococci. J Clin Immunol 10:273–281.[Medline]

This article has been cited by other articles:

				F.X. Lu and R.S. Jacobson Oral Mucosal Immunity and HIV/SIV Infection J. Dent. Res., March 1, 2007; 86(3): 216 - 226. [Abstract] [Full Text] [PDF]

				A. Carapelli, M. Regoli, C. Nicoletti, L. Ermini, L. Fonzi, and E. Bertelli Rabbit Tonsil-associated M-cells Express Cytokeratin 20 and Take Up Particulate Antigen J. Histochem. Cytochem., October 1, 2004; 52(10): 1323 - 1332. [Abstract] [Full Text] [PDF]

This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (17) Citing Articles via Google Scholar Google Scholar Articles by Childers, N.K. Articles by Michalek, S.M. Search for Related Content PubMed PubMed Citation Articles by Childers, N.K. Articles by Michalek, S.M.