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Previous Article | Next Article 
Infection and Immunity, February 1999, p. 618-623, Vol. 67, No. 2
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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
Noel K.
Childers,1,*
Giang
Tong,1
Stephen
Mitchell,1
Katharine
Kirk,2
Michael W.
Russell,3 and
Suzanne
M.
Michalek3
Department of Pediatric Dentistry, School of
Dentistry,1
Department of Biostatistics,
School of Public Health,2 and
Department of Microbiology,3 University
of Alabama at Birmingham, Birmingham, Alabama
Received 17 August 1998/Returned for modification 15 October
1998/Accepted 23 November 1998
 |
ABSTRACT |
Recent attention to mucosal immunization strategies has been
focused on the nasal route for vaccine delivery. This study was designed to determine the effectiveness of a liposome-protein vaccine
compared to that of a protein-only vaccine in inducing immune responses
in humans. Healthy subjects were randomly assigned to two groups and
immunized intranasally with a crude antigen preparation rich in
glucosyltransferase (C-GTF) from Streptococcus mutans,
alone or in liposomes. Parotid saliva, nasal wash, and serum were
collected prior to and at weekly intervals following immunization and
were analyzed for anti-C-GTF activity by enzyme-linked immunosorbent
assay. The levels of immunoglobulin A (IgA) anti-C-GTF activity in the
nasal wash from both groups after immunization increased to a mean peak
of fivefold over the baseline level on day 28. Salivary IgA anti-C-GTF
responses were induced to a lesser extent. IgG and IgA anti-C-GTF
responses in serum were detected on day 14. The IgA responses were
predominantly of the IgA1 subclass. These results show that C-GTF
vaccines were more effective in inducing a local secretory IgA antibody
response than a salivary or serum response when they were given
intranasally. The IgA1 anti-C-GTF response in nasal wash samples for
liposomal antigen versus antigen only was the only response which was
significantly different (P < 0.04). This suggests
that the form of the antigen affects the magnitude of the local mucosal
response but not that of a disseminated response. These results provide
evidence for the effective use of a nasal protein vaccine in humans for
the induction of mucosal and systemic responses.
| |
INTRODUCTION |
Dental caries is considered to be
one of the most prevalent and costly infectious diseases in the world
(16), and despite the reports that this disease is on the
decline in some developed countries (34, 47), it
continues to be a worldwide problem. Secretory immunoglobulin A
(IgA) antibodies in saliva are considered the first line of defense
against pathogens present in the oral cavity, including
Streptococcus mutans, the principal etiologic agent of
dental caries. Therefore, studies aimed at the development of a caries
vaccine have focused on the use of immunization regimens which
stimulate the induction of IgA responses in saliva.
Oral administration of a vaccine results in the induction of IgA
responses in various secretions, including saliva, via the common
mucosal immune system (7). Studies in humans have shown that
oral immunization with antigens from S. mutans results in mucosal IgA responses (8, 11, 43); however, the magnitude of
the immune responses was shown to be low and their persistence limited.
Recently, interest has been directed toward determining the importance
of nasal immunization as a route for inducing mucosal responses,
especially in the upper respiratory tract and oral cavity. The human
nasal mucosa contains an abundance of IgA-secreting plasma cells
(predominantly IgA1) which may originate in bronchus-associated lymphoid tissue or tonsils (5). The human nasal mucosa also contains T lymphocytes (48) and HLA-DR-expressing dendritic and epithelial cells (42), a fact which provides evidence
that it may be an inductive site for responses in nasal (and oral) secretions. In this regard, intranasal (i.n.) immunization against respiratory pathogens (e.g., influenza and parainfluenza viruses) in
humans results in antibody responses in nasal secretions and serum
(12, 32). We have shown that i.n. immunization of
humans with a crude preparation of an S. mutans antigen
preparation rich in glucosyltransferase (C-GTF) in liposomes
resulted in anti-C-GTF responses in nasal wash and saliva
(9).
The present study compared the effectiveness of a liposomal preparation
of S. mutans antigen to that of the free antigen in inducing
mucosal and systemic immune responses after i.n. immunization of humans
in a double-blind study.
| |
MATERIALS AND METHODS |
Bacteria, media, and reagents.
The C-GTF preparation used as
the antigen in this study was derived from S. mutans GS-5 (a
serotype c isolate, obtained from F. Macrina, Virginia Commonwealth
University, Richmond, Va.) and was grown in streptococcal defined
medium (J.R.H. Biosciences, Lenexa, Kans.) (45).
The components used for production of liposomes consisted of
D,L-
-dipalmitoyl phosphatidylcholine,
cholesterol, and dicetylphosphate (Sigma Chemical Company, St. Louis,
Mo.). Liposomes were prepared by sonication of aqueous antigen
suspensions and membrane filtration as previously reported
(9).
Immunological reagents used for enzyme-linked immunosorbent
assay (ELISA) analysis consisted of biotinylated goat anti-human IgA, IgM, and IgG (Biosource Inc., Burlingame, Calif.); unlabeled rabbit or goat anti-human IgA, IgM, and IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, Pa.); and mouse monoclonal IgG anti-human IgA1 and IgA2 (Accurate Chemical & Scientific Corp., Westbury, N.Y.). Biotinylated goat anti-mouse IgG (Southern
Biotechnology Associates Inc., Birmingham, Ala.) was used as the
detecting antibody for the IgA subclass analyses. Fetal calf serum
(Flow Laboratories Inc., McLean, Va.) was used as the blocking reagent
in the ELISA.
Antigen.
C-GTF used for immunization and ELISA was derived
as previously reported (9). Briefly, following growth of
S. mutans GS-5 in a 400-liter broth culture, cells were
removed by centrifugation, the culture supernatant was concentrated by
using a PLGC Pelicon cassette system (10,000 MW cutoff; Millipore Inc.,
Bedford, Mass.), and proteins were precipitated from the supernatant
with 60% saturated ammonium sulfate. The purity, immunogenicity, and
biologic activity of the resulting C-GTF preparation were determined by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
Western blot analysis with antisera to purified cloned glucan binding domain of GTF-I (24) and to purified antigen I/II (AgI/II)
(raised against purified S. mutans AgI/II [IB162])
(39), and periodic acid-Schiff (PAS) staining following
incubation with 10% sucrose, as previously reported (11).
Purified AgI/II was derived from S. mutans IB162 as
previously described (39).
Experimental design for human study.
Twenty-one healthy
adult volunteers ranging in age from 20 to 45 years were recruited to
participate in this study. In compliance with guidelines established by
the University of Alabama at Birmingham (UAB) Institutional Review
Board, informed consent was obtained from the subjects. All but three
subjects (assigned numbers 5, 16, and 20) had previous experience with
dental caries, although none had active or recent carious lesions prior
to or during this study. One subject reported having had a
tonsillectomy (subject 5), one had had an adenoidectomy (subject 18),
and two had had a tonsillectomy and adenoidectomy (subjects 12 and 17)
during childhood.
The 21 subjects were randomly assigned to group A (liposomal C-GTF;
n = 11) or group B (free C-GTF; n = 10). The subjects were immunized i.n. twice (day 0 and day 7) with
the designated preparation by having 68 µl deposited in each nostril
(total dose of C-GTF, 250 µg) while the subject was reclined. Both
the subject and the clinician were blind as to the group assignment.
Unstimulated parotid saliva, nasal wash, and serum samples were
collected weekly for 3 weeks before (baseline) and then weekly for 8 weeks following the initial immunization for analysis of anti-C-GTF
antibody activity. Parotid saliva samples were obtained by using
Schaeffer cups (41). Nasal wash was obtained by depositing
1.5 ml of sterile saline into each nostril while the subject was
reclined and allowing it to remain in the nostril for approximately
10 s. The subject was then instructed to sit up, and the nasal
wash solution was allowed to drain into a specimen cup. Saliva and
nasal wash samples were immediately clarified by centrifugation at
14,000 × g in an Eppendorf centrifuge. Serum was
obtained from centrifuged (14,000 × g) blood collected
by finger stick in Microvette tubes with clotting activator (Sarstedt,
Numbrecht, Germany). All samples were aliquoted and frozen at 
70°C
until used for ELISA.
Antibody analysis.
An ELISA was used to determine the levels
of total immunoglobulin and the relative concentrations of antibodies
to S. mutans C-GTF as previously described (11).
Briefly, polyvinylchloride microtiter plates (Dynatech Laboratories
Inc., Chantilly, Va.) were coated overnight at room temperature with
goat or rabbit anti-human IgA, IgM, IgG, or C-GTF diluted in
phosphate-buffered saline. Optimal dilutions of saliva, nasal wash, or
serum (50 µl) in duplicates of four to eight twofold dilutions were
added to designated wells of the microtiter plates. A human serum pool of known isotype concentrations (Dade Moni-Trol; Baxter Diagnostic Inc., Deerfield, Ill.) and purified human IgA1 or IgA2 myeloma protein
(provided by J. Mestecky, UAB) were used as the total immunoglobulin
standards. Following sample incubation, biotin-conjugated goat
antiserum to human IgA, IgM, or IgG (Biosource) or mouse monoclonal
anti-human IgA1 or IgA2 (Accurate) was added to the appropriate wells.
Color development was accomplished with streptavidin-peroxidase conjugate (Southern Biotechnology Associates), followed by ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)] substrate (Sigma)
in citrate buffer and recorded at 414 nm. A four-parameter curve
fitting program (Softmax; Molecular Devices) was used to construct reference curves for each ELISA plate from optical density readings of the standard plasma pool of known immunoglobulin
concentration. Plasma results were reported in nanograms of anti-C-GTF
antibody per ml, while saliva and nasal wash results were converted to a ratio of anti-C-GTF antibody per total isotype or subclass antibody concentration to normalize for variation in total immunoglobulin content in the samples. For each subject, results obtained from the
three baseline samples were averaged for comparison to postimmunization anti-C-GTF responses (reported as a percent increase over baseline activity for each subject).
Statistics.
A mixed-model analysis was used for comparison
of antibody activity. This analysis considers group (liposomal versus
free antigen), time (e.g., preimmunization versus postimmunization), and group times time to be fixed effects and subject-to-subject variation and its interactions to be random effects. Due to the heterogeneity of variances and right-skewness for the original variables, their common logarithms were analyzed. Significant differences were determined to be P values less than 0.05.
| |
RESULTS |
All 21 individuals completed the study, and none reported any side
effects during or after i.n. immunization.
Vaccine characterization.
S. mutans GS-5 grown in 400 liters of chemically defined medium yielded over 1 g of
proteinaceous material. The relative purity and biological activity of
the C-GTF preparation were confirmed by SDS-PAGE and PAS
staining, respectively (Fig. 1). The
Coomassie blue-stained gel revealed a broad major band at
approximately 161 kDa for C-GTF and a 193-kDa band for AgI/II. PAS
staining following 10% sucrose incubation resulted in a band
corresponding with putative enzymatically active GTF at 161 kDa and
smaller molecular mass moieties (approximately 109 and 86 kDa) also
producing insoluble carbohydrate polymers as previously reported
(10). No activity was seen with purified AgI/II.
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FIG. 1.
SDS-PAGE of C-GTF and purified AgI/II from S. mutans separated on 7% resolving gel. Lanes 1 to 3 are a
Coomassie blue staining of the molecular mass standard (lane 1), C-GTF
(lane 2) and purified AgI/II (lane 3). Lanes 4 and 5 are a PAS staining
of C-GTF (lane 4) and purified AgI/II (lane 5) after incubation with
10% sucrose. The numbers to the left, in kilodaltons, are molecular
mass markers.
|
|
The C-GTF was further characterized by Western blot analysis with
antiserum to purified AgI/II and to cloned glucan binding region of
GTF-I (Fig. 2). Both antisera reacted
with the C-GTF preparation, detecting bands in the molecular mass range
of 150 to 165 kDa. The antisera to AgI/II but not those to GTF reacted with the AgI/II preparation. These results indicate the presence of GTF and a truncated AgI/II protein in the C-GTF preparation.
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FIG. 2.
Western blot analysis of C-GTF (lanes 1 and 3) and
purified AgI/II (lanes 2 and 4) with polyclonal antiserum raised
against purified AgI/II (lanes 1 and 2) or cloned glucan binding domain
of GTF-I (lanes 3 and 4). Lane 5 is the molecular mass standard. The
numbers to the right, in kilodaltons, are molecular mass markers.
|
|
Nasal wash response.
A nasal IgA1 anti-C-GTF response was
detected in 19 of 21 subjects (i.e., the response was at least twofold
over baseline at some time points following immunization). Individuals
given liposomes containing C-GTF (group A) had a higher response
throughout the experiment than those given free C-GTF (group B) (Fig.
3). The difference in the response was
significant (P < 0.04) on day 28.
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FIG. 3.
Nasal IgA1 anti-C-GTF activity as a percentage of total
IgA1 antibody activity before and after i.n. immunization. Values are
the ratio of C-GTF-specific/total immunoglobulin for samples collected
before and after i.n. immunization with liposomal C-GTF (group A
[ ]) or free C-GTF (group B [ ]). *, mean difference
between group A and group B values is significantly different
(P < 0.04) on day 28.
|
|
Since no significant difference was seen between the two groups for all
other analyses (time points, including serum and saliva), the data from
groups A and B were combined. Preimmunization variability was found to
be comparatively low when related to postimmunization data for the
nasal wash IgA anti-C-GTF responses (Fig.
4a). Mixed-model analysis resulted in a
highly significant (P < 0.0001) day effect, which
corresponded to values obtained for the three preimmunization samples
and that from day 7, in contrast to the values obtained with samples
collected at the seven remaining time points (postresponse) with a mean
peak of a fivefold increase in the ratio of IgA anti-C-GTF/total over
baseline occurring on day 28.
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FIG. 4.
(a) Nasal IgA anti-C-GTF activity as a percentage of
total antibody activity before and after i.n. immunization. Values are
the mean ratio of anti-C-GTF-specific/total immunoglobulin activity
(plus standard deviation) in nasal wash samples collected from human
subjects before and after i.n. immunization with C-GTF (combined data
from liposomal C-GTF and free C-GTF groups). (b) Nasal IgA1 ( ) and
IgA2 ( ) anti-C-GTF activity as a percentage of total antibody
activity after i.n. immunization. Values are the mean percent increase
in anti-C-GTF/total immunoglobulin activity in nasal wash samples (over
three preimmunization samples) collected from human subjects after i.n.
immunization with C-GTF (combined data from liposomal C-GTF and
free C-GTF groups).
|
|
A highly significant (P < 0.0001) IgA subclass day
(i.e., response) effect was also observed (Fig. 4b). The peak (day 28) percent increase in IgA1 compared to the IgA2 response was not only
higher (fourfold versus twofold, respectively), but the magnitude of
IgA1 (total and specific antibody activity; Table
1) was also ~2 to 3 times that of IgA2.
Saliva response.
The salivary IgA anti-C-GTF activity in 9 of
21 subjects was at least twofold over baseline with a mean peak of a
73% IgA anti-C-GTF increase over baseline occurring on day 21. Although variable and relatively small, the change in the mean level of salivary IgA anti-C-GTF/total for four preresponse samples, compared to
seven postresponse samples, was significantly different by mixed-model analysis (P < 0.0001; Fig.
5).
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FIG. 5.
Salivary IgA anti-C-GTF activity as a percentage of
total IgA antibody activity before and after i.n. immunization. Values
are the mean ratio of C-GTF-specific/total immunoglobulin (plus
standard deviation) in anti-C-GTF activity in parotid saliva samples
collected from human subjects before and after i.n. immunization with
C-GTF (combined data from liposomal C-GTF and free C-GTF groups).
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|
The mean ratio of IgA1 anti-C-GTF/total response was a greater percent
increase over baseline level than that of IgA2 (82 and 23%,
respectively). Furthermore, the magnitudes of IgA1 and IgA2 antibody
responses in the samples were similar, i.e., the ratio of IgA1/IgA2 was
1.2 to 1.4 (Table 1).
Serum response.
An IgA1 response in serum was detected in 13 of 21 subjects with a mean increase of 1.5- to 2-fold over baseline
from day 21 to 56 following immunization (Fig.
6). IgA1 responses persisted through the
termination of the experiment (day 56). No significant IgA2 antibody
response was found in serum. The mean IgG response was of a lower
magnitude (~40% mean increase over baseline; Fig. 6), with only 4 of
21 subjects showing an increase of more than twofold over baseline
anti-C-GTF activity.
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FIG. 6.
Serum IgA1 ( ) and IgG ( ) anti-C-GTF responses
after i.n. immunization with liposomal C-GTF. Values are the percent
increase in anti-C-GTF activity in serum samples (over three
preimmunization samples) collected from human subjects after i.n.
immunization with C-GTF (combined data from liposomal C-GTF and free
C-GTF groups).
|
|
Responses to AgI/II.
An analysis by ELISA of saliva, serum,
and nasal wash collected from individuals immunized with liposomal
C-GTF preparation by using purified AgI/II as the coating antigen
revealed response patterns similar to those obtained with the
homologous antigen C-GTF (data not shown).
| |
DISCUSSION |
Historically, studies to promote the induction of salivary immune
responses in humans by mucosal immunization have mainly concentrated on
administering antigen via the gastric route (31). The
rationale behind this approach was that oral immunization results in
mucosal responses by way of the common mucosal immune system, i.e.,
antigen-sensitized lymphoid cells in the gut-associated lymphoid tissue
(IgA inductive sites) are disseminated to various mucosal effector
sites. Recent evidence, however, suggests that compartmentalization may
exist in the mucosal immune system, whereby the response pattern seen
at various mucosal effector sites may differ according to the route of
mucosal immunization (30, 33). As a result of this
information, much interest has been placed on determining the
importance of Waldeyer's ring as an inductive site for mucosal
responses, especially for responses in the upper respiratory tract and
oral cavity. Numerous reports on i.n. immunizations with antigens
(19, 21, 22, 25, 29, 38, 40, 44, 49) or with liposomal
antigens (1-3, 6, 14, 15, 20) involving various animal
models have provided encouraging results, indicating that this type of
immunization increases antigen-specific antibody responses in pulmonary
and oral secretions.
The present study was initiated to investigate the effectiveness of
immunizing humans via the i.n. route with S. mutans
antigens, either incorporated into liposomes or free, in inducing
salivary IgA responses. The present findings expanded our preliminary
results (9) regarding the immunogenicity of C-GTF when given
i.n. to five human subjects. The inclusion of a larger sample size
greatly enhanced the statistical relevance of our findings. In this
regard, we observed significantly higher mean postimmunization
IgA anti-C-GTF activity in nasal wash, parotid saliva, and serum than
that observed preimmunization.
This study also involved a blind clinical test of two forms of the
vaccine antigen, i.e., liposomal and free antigen. Subjects given the
liposomal C-GTF by the i.n. route had significantly higher nasal wash
IgA1 immune response levels than volunteers given the free antigen.
However, the finding that no significant difference in responses was
detected in other samples suggests that soluble C-GTF antigen was as
effective as liposomal C-GTF in inducing salivary and serum responses
when given by the i.n. route. Alternatively, it is recognized that
sample sizes are small in this study and that power may not have been
sufficient to detect small differences.
Previous studies (9, 11) have shown that the culture
supernatant of S. mutans GS-5 grown in chemically
defined medium is enriched for GTF. An ammonium sulfate precipitation
of this preparation results in a fraction which predominantly consists of a 165-kDa protein, determined by SDS-PAGE, is enzymatically active
in the presence of sucrose, and is immunogenic when given to animals
and humans. Some strains of S. mutans, including GS-5, are known to produce a truncated AgI/II surface protein (~155 kDa)
that is released into the culture medium (35). Biochemical and immunological analysis of the C-GTF antigen preparation indicate the presence of both GTF and truncated AgI/II. These analyses also
indicated the presence of other antigens. The two additional bands
indicating insoluble carbohydrates in the PAS stain of gels following
incubation with sucrose may represent fructosyltransferase or glucan
binding proteins. Furthermore, minor bands identified in Western blots
may illustrate other antigens or breakdown products in the C-GTF
preparation. Immunization of humans by the i.n. route with C-GTF
resulted in the induction of immune responses to the immunogen and
native AgI/II. Further studies are required to define the dynamics of
the responses to purified GTF and AgI/II, as well as other antigens in
the C-GTF which may have contributed to the immune responses detected,
in different body fluids and the effectiveness of these responses in
inhibiting S. mutans infection. Since GTF and AgI/II
are considered virulence antigens of S. mutans, which are involved in two different stages of the pathogenesis of dental caries, i.e., sucrose-independent reversible attachment mediated by
AgI/II and sucrose-dependent irreversible attachment mediated by GTF
(36), the induction of immune responses to C-GTF could act
against both stages. Therefore, the potential for an additive or
synergistic protective benefit of the bivalency (or potentially multivalency) of the C-GTF used in this study is attractive in the
continued development and evaluation of caries vaccines for use in humans.
An overall goal of our studies is to determine a mucosal route of
immunization with an S. mutans vaccine that induces
optimal, effective salivary immune responses. Although a significant
increase was seen in the salivary IgA response after i.n. immunization, the response was lower than that seen in nasal wash and serum. It is
possible that the salivary response induced would be effective in
protecting against S. mutans infection, as suggested by
the results of experimental animal studies (31).
Nevertheless, further studies are needed to identify a mucosal
immunization route, dosage, antigen form, adjuvant, and timing schedule
for further enhancing the magnitude and longevity of the salivary
response. These studies may involve additional testing of i.n.
immunization strategies of other routes.
Since i.n. immunization resulted in primarily a nasal response,
there may exist a mucosal IgA inductive site which preferentially promotes a salivary response. Fukuizumi and coworkers
(17) have presented data that indicate that tonsils may play
a role in the selective induction of oral responses. In that study,
rabbits were immunized with sheep erythrocytes by the i.n. or tonsillar (pharyngeal) route. Antibody activity was seen in nasal washes after
nasal immunization, while salivary responses resulted from tonsillar
immunization (antigens topically applied via the oral cavity). In a
recent study these investigators, using S. mutans whole
cell antigen, also reported the induction of a salivary response
following tonsillar immunization (18).
The tonsils are a potential induction site for responses
within the oral cavity. Waldeyer's ring (consisting of palatine, lingual, and nasopharyngeal [adenoids] tonsils), located at the proximal end 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 (4, 27). The unique architecture of the tonsils
resembles that of lymph nodes (4) and gut-associated lymphoid tissue (23), in that they have antigen-presenting
cells, T and B lymphocytes, IgG- and IgA-containing plasma cells in
characteristic regions (4, 23, 46), and deep branched crypts
which increase the surface area for trapping environmental materials
(4). Supporting evidence for the importance of tonsils in
local immune responses include (i) secretion of polymeric IgA in
cultured tonsillar cells (28); (ii) the predominance of
IgA1, typical of the upper respiratory and digestive tracts (13,
26); and (iii) reduced nasopharyngeal antibody responses to
perorally administered live poliovirus in tonsillectomized children and
their decreased nasopharyngeal resistance due to diminished secretory
IgA levels (37). Although the tonsils were not directly
immunized in our study, 5 of the 21 subjects had a history of tonsil
surgery. The subjects involved in this study who had had a
tonsillectomy and/or adenoidectomy had response patterns and levels of
IgA in saliva and nasal wash which were similar to or lower than the
average in the other subjects. A more comprehensively designed study
comparing the immune responses in individuals with and without a
history of tonsil surgery is necessary to determine if responses differ
in subjects who have had a tonsillectomy. Alternatively, to determine
if tonsils are an IgA induction site for salivary responses, a study in
which antigen is topically applied to tonsil tissue is needed and will help determine the potential for tonsils to induce more optimal oral responses.
i.n. immunization of humans with liposomal or free C-GTF antigen
of S. mutans resulted in immune responses in nasal
secretions, parotid saliva, and serum. The liposomal antigen vaccine
induced higher nasal but similar salivary IgA responses, compared
to responses induced with the free antigen vaccine. Additional studies
are needed to identify ways of inducing predominantly salivary IgA responses, in order to design a more effective approach to prevent oral
diseases, e.g., dental caries.
| |
ACKNOWLEDGMENTS |
We thank Jiri Mestecky for myeloma proteins and Christina
Jespersgaard from UAB Department of Microbiology for purified cloned glucan binding domain of GTF-I. We also wish to thank Rosie Turner for
secretarial help.
This work was supported in part by NIH grants DE09846, DE09081,
DE08182, and DE08228 and grants DE06746 from the NIDR.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pediatric Dentistry, School of Dentistry, Room 312, University of
Alabama at Birmingham, Birmingham, AL 35294-0007. Phone: (205)
934-3230. Fax: (205) 975-5737. E-mail: nkc{at}uab.edu.
Editor:
V. A. Fischetti
| |
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Infection and Immunity, February 1999, p. 618-623, Vol. 67, No. 2
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
