| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Immunobiology Vaccine Center, Departments of Oral Biology and Microbiology, University of Alabama Medical Center, Birmingham, AL 35294; and
Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in CLN at both the protein and mRNA
levels. Importantly, significant numbers of mice intranasally immunized
with PspA plus mCT S61F were protected from lethal challenge with
capsular serotype 3 Streptococcus pneumoniae A66. These
results show that intranasal administration of PspA together with mCT
S61F is an effective mucosal vaccine against pneumococcal infection and
induces CD4+ Th2-type cells, which provide help for both
mucosal and systemic Ab responses. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Protein vaccines have been considered as alternative approaches to induce protective immune response in infants and children to protect from pneumococcal infection. In particular, pneumococcal surface protein A (PspA),3 which is a surface protein of S. pneumoniae and is highly immunogenic, is considered to be a promising vaccine candidate for combating pneumococcal infection (5, 6, 7). Not only is it a major virulence factor common to all isolates of S. pneumoniae (5, 6, 7, 8, 9, 10), but mAbs to it have been shown to protect mice from fatal pneumococcal challenge (11). Immunization studies using PspA have also shown that full-length PspA and the isolated N-terminal half of PspA both induce protective immunity (10, 12). Furthermore, recombinant bacillus Calmette-Guérin expressing PspA as a secreted protein or as a chimeric exported membrane-associated lipoprotein can protect against systemic challenge with different strains of S. pneumoniae (13). Thus, PspA appears to be the most attractive candidate Ag for future development of a protein-based vaccine to protect from pneumococcal infections.
Recently, much effort has been focused on inducing immune responses in mucosal tissues since pneumococcal infection is acquired through the mucus membranes of the upper respiratory tract. It has been shown that intranasal immunization with PspA plus cholera toxin B subunit (CT-B) as an adjuvant induces mucosal IgA Ab responses and provides protection against carriage of S. pneumoniae (14). In a previous study, we also demonstrated that oral administration of PspA with cholera toxin (CT) as adjuvant induced PspA-specific mucosal IgA Abs as well as protective immunity (15). Although the combination of PspA with the adjuvant CT or CT-B was then shown to be effective against pneumococcal infection, CT, an enterotoxin that causes clinical manifestations of cholera, is unsuitable for use in humans.
In order to eliminate the diarrheagenicity but retain the adjuvanticity of CT, we generated two mutants of CT (mCT) (S61F and E112K), which harbor single amino acid substitutions in the ADP-ribosyltransferase active center that render them enzymatically inactive and thus nontoxic (16). Interestingly, these mutants support Ag-specific immune responses when administered parenterally (16). Furthermore, we have found that mCT S61F acts as a mucosal adjuvant by inducing CD4+ Th2 cells secreting IL-4, IL-5, IL-6, and IL-10, which provide effective help for Ag-specific secretory IgA as well as serum IgG1, IgE, and IgA Ab responses (17).
In this study, we have evaluated the efficacy of using a mucosal vaccine to prevent pneumococcal infection. The results obtained in this study suggest that nasal PspA plus mCT S61F would be a practical and effective vaccine candidate for induction of Ag-specific Ab responses in both mucosal and systemic compartments.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C57BL/6 mice were purchased from The Jackson Laboratory Animal Resources Center (Bar Harbor, ME) and were maintained and bred in this facility under pathogen-free conditions in microisolator cages. All mice were provided sterile food and water ad libitum and were used in this study at 8 to 12 wk of age.
PspA and adjuvants
Full length native PspA was prepared from R36A pneumococci (10). Briefly, supernatants were prepared from pneumococci grown in defined medium in which choline had been replaced with ethanolamine. Pneumococci grown under these conditions release PspA into the medium. This PspA was isolated by passing the PspA-containing supernatants over a choline-Sepharose column. After washing the column with tris-acetate (TA) buffer, the PspA was eluted with a 2% choline in TA buffer.
Escherichia coli containing the plasmid for the mCT S61F gene were grown in LB medium (NaCl, 10 mg/ml; yeast extract, 5 mg/ml; tryptone, 10 mg/ml) with 100 µg/ml of ampicillin. The mCT S61F was purified using a D-galactose-immobilized column (Piece, Rockford, IL) from a cell suspension prepared by sonication of the bacteria, as described previously (16). The purity of mCT S61F was assessed by SDS/PAGE and no contaminating protein bands were noted (17). Native CT (nCT) was purchased from List Biologic Laboratories (Campbell, CA).
Immunization and sample collection
Mice were intranasally immunized on days 0, 7, and 14 with a 10-µl aliquot (5 µl per nostril) containing 100 ng of PspA alone or combined with either 5 µg of mCT S61F or 0.5 µg of nCT (17). Saliva was obtained from mice following i.p. injection with 100 µl of 1 mg/ml pilocarpine (Sigma, St. Louis, MO). Nasal washes were collected by gently flushing the nasal passage with 100 µl of sterile PBS.
Detection of Ag-specific Ab isotype and IgG subclass responses
Ab titers in serum, saliva, and nasal wash were determined by
ELISA as described previously (18). Briefly, plates were
coated with PspA (100 ng/ml) and blocked with 10% goat serum in PBS.
After washing, serial dilutions of serum or fecal extracts were added
in duplicate. Following incubation, the plates were washed and
peroxidase-labeled goat anti-mouse µ, , or 
heavy
chain-specific Abs (Southern Biotechnology Associates, Birmingham, AL)
were added to appropriate wells. Finally,
2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) with
H2O2 (Moss, Pasadena, CA) was added for color
development. For IgG subclass analysis, biotinylated rat monoclonal
anti-mouse l (G1-7.3), 2a (R19-15), 2b (R12-3), or 3
(R40-82) heavy chain-specific Abs (PharMingen) and
streptavidin-conjugated peroxidase were employed. Endpoint titers were
expressed as the reciprocal log2 of the last dilution that
gave an OD at 414 nm of 0.1 greater than background after 15 min of
incubation. In no case did nonimmunized mice give titers greater than
log2 of 5 in sera and log2 of 2 in external
secretions.
Ab-forming cell (AFC) assay
Cervical lymph nodes (CLN) and spleens were aseptically removed
and single cell suspensions were obtained as described
(19). The nasal passage, submandibular gland (SMG), and
lung tissues were carefully excised, teased apart, and dissociated
using collagenase type IV (Sigma) (19, 20). The
mononuclear cells were obtained at the interface of the 40 and 75%
layers of a discontinuous Percoll gradient (Pharmacia). To assess
number of PspA-specific AFCs, an enzyme-linked immunospot (ELISPOT)
assay was performed as previously described (15). Briefly,
96-well nitrocellulose plates (Millititer HA; Millipore, Bedford, MA)
were coated with PspA (100 ng/ml), incubated, and then plates were
washed and blocked with 10% goat serum. The blocking solution was
discarded, cells at various dilutions were added, and the cell
suspensions were incubated for 4 h at 37°C in 5%
CO2 in moist air. The detection Ab consisted of goat
anti-mouse or heavy chain-specific, horseradish
peroxidase-conjugated Abs (Southern Biotechnology Associates).
Following overnight incubation, plates were washed with PBS and
developed by addition of 3-amino-9-ethylcarbazole dissolved in 0.1 M
sodium acetate buffer containing H2O2 (Moss) to
each well. Plates were incubated at room temperature for 15 to 20
min and washed with water, and AFCs were counted with the aid of a
stereomicroscope.
PspA-specific CD4+ T cell responses
CD4+ T cells from CLN cell suspensions were purified by use of the magnetic-activated cell sorter system (Miltenyi Biotec, Sunnyvale, CA). Cells were added to a nylon wool column (Polysciences, Warrington, PA) and incubated at 37°C for 1 h to remove adherent cells. The enriched T cell populations were then incubated with biotinylated anti-CD8 (53-6.7), anti-Mac-1 (M1/70), and anti-B220 (RA3-6A2) mAbs followed by streptavidin-conjugated microbeads before being passed through the magnetic column. Two cycles of the above procedures yielded CD4+ T cell preparations that were >98% pure. Purified CLN CD4+ T cells (2 x 106 cells/ml) were cultured with T cell-depleted, irradiated splenic feeder cells (2.5 x 106 cells/ml) from naive mice in complete medium (RPMI 1640, Cellgro Mediatech, Washington, DC) containing 10% FCS, 1% L-glutamine, 50 µM 2-ME, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 40 µg/ml gentamicin. The cultures were supplemented with 10 U/ml of mouse rIL-2 (Genzyme, Cambridge, MA). PspA (1 µg/ml) was added for restimulation of CD4+ T cells. The CD4+ T cell cultures were incubated for 4 days at 37°C in 5% CO2 in air. To measure cell proliferation, 0.5 µCi of [3H]thymidine (DuPont/New England Nuclear Products, Boston, MA) was added to individual culture wells 15 h before termination, and the uptake of counts per minute by dividing cells was determined by scintillation counting.
Quantitative analysis of cytokine-specific mRNA
For evaluation of cytokine-specific mRNA levels, a quantitative
RT-PCR was employed. Total RNA was isolated by the acid guanidinium
thiocyanate phenol chloroform extraction procedure. Cytokine-specific
rDNA for IFN- and IL-4 were used as the internal standards as
described in detail previously with minor modifications (16, 21). Briefly, aliquots of total RNA were subjected to standard
RT. RT products with a series of diluted rDNA internal standards were
amplified by PCR. For quantification, capillary electrophoresis with
the laser-induced fluorescence detection system (CE-LIF) (LIF-P/ACE,
Beckman Instruments, Fullerton, CA) was applied as described previously
(22, 23). An analysis of PCR products was conducted by
using a coated capillary tube in Tris-borate EDTA containing
replaceable linear polyacrylamide and fluorescent intercalator. The
fluorescence content of each cytokine-specific RT-PCR product was
expressed as the peak area of relative fluorescence light units.
Analysis of secreted cytokines
Cytokine levels in culture supernatants were determined by a
cytokine-specific ELISA as described previously (24, 25).
Nunc MaxiSorp Immunoplates (Nunc, Naperville, IL) were coated with
monoclonal anti-IFN- (R4-6A2) or anti-IL-4 (BVD4-1D11) Abs
(PharMingen). After blocking, samples and serial twofold dilutions of
standards were added to duplicate wells and incubated overnight at
4°C. The wells were washed and incubated with biotinylated monoclonal
anti-IFN- (XMG 1.2) or anti-IL-4 (BVD6-24G2). After
incubation, peroxidase-labeled anti-biotin Ab (Vector Laboratories,
Burlingame, CA) was added and developed with ABTS containing
H2O2 (Moss). Standard curves were generated
using mouse rIFN- (Genzyme) and rIL-4 (Endogen, Boston,
MA).
Mouse protection assay
The pneumococcal in vivo protection assay was carried out as described previously using log-phase pneumococci diluted in Ringer’s lactate (26). Four weeks after the last immunization, mice were challenged i.v. with 2.5 x 106 CFU of capsular serotype 3 S. pneumoniae A66. The survival of the mice was monitored for 12 days.
Statistics
The data are expressed as the mean ± SEM and compared using one-tailed Wilcoxon two-sample rank test. The results were analyzed using the Statview II statistical program (Abacus Concepts, Berkeley, CA) adapted for Macintosh computers.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the initial study, we sought to determine if intranasal
administration of PspA with mCT S61F as mucosal adjuvant could induce
PspA-specific Ab responses. Mice intranasally immunized with PspA
plus mCT S61F showed significant PspA-specific serum IgM, IgG, and
IgA Ab responses, which were comparable to those induced by PspA with
nCT as adjuvant. In contrast, Ab responses were not detected after
immunization with PspA alone (Fig. 1
A). As expected,
administration of mCT S61F or nCT alone did not induce PspA-specific Ab
responses that were above the dilution cutoff (log2 of 5)
used in our experiments. Analysis of IgG subclass responses in mice
given PspA plus mCT S61F or nCT revealed that the major subclasses were
IgG1 and IgG2b (Fig. 1A). AFC responses supported the serum
Ab titers and showed that significant numbers of PspA-specific IgG- and
IgA-producing cells were found in the spleens of mice given PspA plus
mCT S61F or nCT as adjuvants. Furthermore, high numbers of IgG and IgA
AFCs were detected in cells isolated from CLN and lung tissues of mice
given PspA with mCT S61F or nCT as mucosal adjuvants (Fig. 1B).
|
).
As expected, nasal delivery of mCT S61F or nCT alone failed to elicit
PspA-specific Ab titers greater than log2 of 2. AFC
analysis confirmed the above results by revealing significant numbers
of PspA-specific IgA AFC in SMG and nasal passages following intranasal
administration of PspA plus mCT S61F or nCT and by showing low AFC
responses in SMG and nasal passages of mice given PspA alone (Fig. 2).
|
Since intranasal immunization with PspA plus mCT S61F induced
PspA-specific Ab responses in both mucosal and systemic compartments,
it was important to establish the nature of the CD4+ T cell
help supporting PspA-specific Ab responses. When CD4+ T
cells from spleen or CLN of mice immunized with PspA plus mCT S61F were
restimulated with PspA in vitro, levels of proliferative responses
induced were identical to those seen with CD4+ T cells from
mice immunized with PspA plus nCT. Essentially no increased
proliferation occurred in spleen or CLN taken from mice given PspA
alone (Fig. 3). These results indicate
that mCT S61F, like nCT, is an effective adjuvant for the induction of
PspA-specific CD4+ T cells in both mucosal and systemic
tissues.
|
(Th1-type)-specific mRNA. In contrast, no
IL-4-specific mRNA was detected in CD4+ T cells from mice
given PspA alone. Furthermore, IFN--specific mRNA was detectable in
mice given PspA alone but in much lesser quantity than that observed in
mice given mCT S61F or nCT (Fig. 4A).
|
production were further analyzed at the protein level.
Increased levels of IL-4 production with low IFN- were seen in
PspA-specific cultures from mice nasally immunized with PspA plus mCT
S61F or nCT, a finding in complete agreement with results of cytokine
analysis at the transcriptional level (Fig. 4
B). As
expected, CLN CD4+ T cells from mice given PspA alone
produced low or undetectable levels of IFN- or IL-4. Taken together,
these studies show that intranasal administration of PspA plus mCT S61F
as adjuvant induces Th2-type cytokine responses to support
PspA-specific mucosal IgA as well as serum IgG1 and IgG2b Ab
responses. Intranasal immunization with PspA plus mCT S61F elicits protective immunity
In the present study, we sought to determine whether intranasal
PspA plus nontoxic mCT S61F, which elicited immune responses to PspA,
were also protective. Mice given PspA plus mCT S61F, PspA plus nCT, or
PspA alone were challenged i.v. with virulent A66 pneumococci. As
expected, intranasal immunization with PspA plus nCT provided
significant protection (p = 0.0011) against a
lethal dose of A66 (Fig. 5). It is
important to note that PspA given intranasally with nontoxic mCT S61F
possessed protective ability (p = 0.0009)
comparable to or even better than that induced by nCT. In contrast, all
mice given PspA alone, mCT S61F alone, or nCT alone were not protected
and died within 3 days (Fig. 5). These findings indicate that Ab
responses elicited by PspA plus mCT S61F were protective against
pneumococcal infection.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
CT and purified CT-B have been used widely as adjuvants for mucosal
immunization (14, 15, 18, 28, 29, 30, 31, 32, 33, 34), and our results
indicate that intranasal administration of nCT possesses adjuvant
effects to PspA and induces PspA-specific mucosal IgA, as well as serum
IgM, IgG, and IgA Ab responses. However, despite these beneficial
attributes, CT is unsuitable for humans since it causes severe diarrhea
(35). CT consists of one A subunit (CT-A) and five B
subunits (35). CT-B selectively binds GM1 cell surface
receptors and promotes the entry of CT-A. Following entry into cells,
CT-A catalyzes ADP-ribosylation of Gs, resulting in elevated
intracellular cyclic AMP levels, which in epithelial cells cause
secretion of water and chloride ions into the small intestine with a
characteristic watery diarrhea (36, 37). Thus, in order to
develop an adjuvant suitable for use with the pneumococcal vaccine for
humans, ADP-ribosytransferase activity of CT-A must be eliminated. The
studies presented here again demonstrate that mutant CT is an effective
mucosal adjuvant for intranasal immunization, and that when given with
PspA, it facilitates the development of protective levels of Ab to
PspA. The results presented here are the first to show that a nontoxic
mutant of CT, designated S61F and carrying a single amino acid
substitution in the A subunit, induces Ab responses in both serum and
mucosal secretions to coadministered PspA. These findings suggest that
mCT S61F may be a more promising mucosal adjuvant for PspA than
nCT.
Intranasally coadministered mCT S61F, which was shown to lack ADP-ribosyltransferase activity and diarrheagenicity (16), possessed adjuvant effects for PspA. Earlier studies had suggested that the adjuvanticity of CT and that of the related E. coli heat-labile toxin (LT) were closely associated with ADP-ribosyltransferase activity (38). However, our previous studies have shown that two mutants of CT (S61F and E112K), which lack ADP-ribosyltransferase activity, still supported Ag-specific immune responses (16, 17). Furthermore, several groups have reported that single amino acid substitution mutants of LT (R7K, S63K, and R192G), which lack toxicity, retain their adjuvant properties (39, 40, 41). These studies suggest that ADP-ribosyltransferase activity is not required for the adjuvant effects of CT and LT. However, it should be noted that although mCT S61F possesses adjuvanticity in the absence of ADP-ribosyltransferase activity, 10-fold higher doses of mCT S61F were required to elicit PspA-specific Ab responses comparable with nCT, implying that adjuvanticity of CT may be derived from two or more mechanisms and that ADP-ribosyltransferase activity may also contribute to the adjuvanticity of CT and derivatives. Studies are underway to elucidate these additional steps.
Although past studies have demonstrated that CT and purified CT-B
possess adjuvant effects for PspA and induce PspA-specific Ab responses
in serum and mucosal secretions (14, 15), the nature of
helper CD4+ T cells for these Ab responses has not been
established. In this study, we have shown that PspA plus mCT S61F
induced PspA-specific CD4+ T cells in CLN and spleen when
given intranasally. Furthermore, analysis of cytokine patterns produced
by CD4+ T cells revealed significant IL-4 synthesis with
minimal IFN- synthesis, indicating that PspA with mCT S61F elicits
PspA-specific CD4+ Th2-type cells in both mucosal and
systemic tissues. Previous studies by our group support this finding by
showing that mCT, like nCT, induces significant Th2-type cytokine
responses with subsequent serum IgG1, IgG2b, IgE, and IgA, and mucosal
IgA Abs to coadministered Ag when given intranasally (17)
or orally (unpublished observation). Taken together, these findings
indicate that PspA plus mCT S61F as mucosal adjuvant generates
PspA-specific Th2-type responses that account for the induction of
IgG1, IgG2b, and IgA Ab responses in serum, and IgA Abs in external
secretions of the respiratory tract.
The cellular and molecular basis for induction of Ab responses by the mucosal adjuvant CT has not yet been elucidated. A previous study has shown that B7-1 (CD80) and B7-2 (CD86) expression on B cells is enhanced by the administration of CT or a fusion protein retaining an intact A1 subunit and so exhibiting ADP-ribosyltransferase enzyme activity (42). Other studies have shown that CT enhances B7-2 but not B7-1 on macrophages and that macrophages treated with CT enhance proliferative responses of T cells activated by anti-CD3 or alloantigens (43). Both studies suggested that CT adjuvanticity was associated with B7 cosignaling and that ADP-ribosyltransferase enzyme activity was required for the induction of B7 expression. However, our separate study has indicated that mCT, which completely lacks ADP-ribosyltransferase activity, induced both B7-1 and B7-2 expression on Peyer’s patch B cells and macrophages (M.Y., unpublished observation). Furthermore, our present and past studies demonstrate that mCT induces Ag-specific serum IgG and mucosal IgA Ab responses to coadministered proteins (17). Based upon our studies, we conclude that ADP-ribosyltransferase activity can be separated from the adjuvant properties of CT.
The immunization protocol used in this study was designed to induce significant PspA-specific Ab responses in mucosal secretions and serum. We chose intranasal administration as the delivery route because it offered several advantages over oral immunization. First, intranasal administered Ag interacts directly with IgA-inductive sites termed nasal-associated lymphoreticular tissue. Second, lower doses of vaccine Ag are required because this mode of delivery does not subject the Ag to the enzyme and acids of the gastrointestinal tract and so minimizes Ag loss through the process of digestion. Only 100 ng of PspA were required to generate an Ab response equivalent to that elicited by 7.5 µg of orally administered PspA (15). Third, it has been suggested that since intranasal immunization induces lower IgE Ab responses than those induced by oral immunization, this immunization regimen may have less risk for anaphylactic reactions (17).
In summary, the present study has provided evidence that intranasal immunization with a vaccine containing PspA plus mCT S61F elicited PspA-specific CD4+ Th2 cells in both mucosal and systemic tissues with subsequent IgA Ab responses at the mucosal surfaces of the respiratory tract, and IgG and IgA Abs in serum. Furthermore, PspA-specific responses induced by PspA plus mCT S61F provided protective immunity against pneumococcal infection. The combination of PspA and mCT provides a very effective means of eliciting protective levels of Ab to PspA, and therefore should be considered as a candidate adjuvant for vaccinating humans against pneumococci.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Jerry R. McGhee, The Immunobiology Vaccine Center and the Department of Microbiology, University of Alabama, 761 Bevill Biomedical Research Building, 845 19th Street South, Birmingham, AL 35294-2170. E-mail address:
3 Abbreviations used in this paper: PspA, pneumococcal surface protein A; CT, cholera toxin; mCT, mutant cholera toxin; nCT, native cholera toxin; CT-B, cholera toxin B subunit; CLN, cervical lymph nodes; SMG, submandibular gland; ELISPOT, enzyme-linked immunospot; AFC, Ab-forming cells; CE-LIF, capillary electrophoresis with the laser-induced fluorescence detection system; CT-A, cholera toxin A subunit; LT, labile toxin; ABTS, 2,2'-azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid.
Received for publication February 2, 1998. Accepted for publication June 5, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-producing T cells, T cell receptor expression, and IgA plasma cells fro mouse salivary gland-associated tissues. J. Immunol. 148:2030.[Abstract]
and ß T cells. Eur. J. Immunol. 25:2743.[Medline]
This article has been cited by other articles:
|
|
 |
|
  F. Momoi, T. Hashizume, T. Kurita-Ochiai, Y. Yuki, H. Kiyono, and M. Yamamoto Nasal Vaccination with the 40-Kilodalton Outer Membrane Protein of Porphyromonas gingivalis and a Nontoxic Chimeric Enterotoxin Adjuvant Induces Long-Term Protective Immunity with Reduced Levels of Immunoglobulin E Antibodies Infect. Immun., June 1, 2008; 76(6): 2777 - 2784. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Hagiwara, Y. I. Kawamura, K. Kataoka, B. Rahima, R. J. Jackson, K. Komase, T. Dohi, P. N. Boyaka, Y. Takeda, H. Kiyono, et al. A Second Generation of Double Mutant Cholera Toxin Adjuvants: Enhanced Immunity without Intracellular Trafficking. J. Immunol., September 1, 2006; 177(5): 3045 - 3054. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. W. van Ginkel, R. J. Jackson, N. Yoshino, Y. Hagiwara, D. J. Metzger, T. D. Connell, H. L. Vu, M. Martin, K. Fujihashi, and J. R. McGhee Enterotoxin-Based Mucosal Adjuvants Alter Antigen Trafficking and Induce Inflammatory Responses in the Nasal Tract Infect. Immun., October 1, 2005; 73(10): 6892 - 6902. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-Y. Ko, H.-J. Ko, W.-S. Chang, S.-H. Park, M.-N. Kweon, and C.-Y. Kang {alpha}-Galactosylceramide Can Act As a Nasal Vaccine Adjuvant Inducing Protective Immune Responses against Viral Infection and Tumor J. Immunol., September 1, 2005; 175(5): 3309 - 3317. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Kobayashi, T. Kohda, K. Kataoka, H. Ihara, S. Kozaki, D. W. Pascual, H. F. Staats, H. Kiyono, J. R. McGhee, and K. Fujihashi A Novel Neurotoxoid Vaccine Prevents Mucosal Botulism J. Immunol., February 15, 2005; 174(4): 2190 - 2195. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. A. Green, Y. Zhang, A. W. Masi, V. Barniak, M. Wetherell, R. P. Smith, M. S. Reddy, and D. Zhu PppA, a Surface-Exposed Protein of Streptococcus pneumoniae, Elicits Cross-Reactive Antibodies That Reduce Colonization in a Murine Intranasal Immunization and Challenge Model Infect. Immun., February 1, 2005; 73(2): 981 - 989. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Yoshino, F. X.-S. Lu, K. Fujihashi, Y. Hagiwara, K. Kataoka, D. Lu, L. Hirst, M. Honda, F. W. van Ginkel, Y. Takeda, et al. A Novel Adjuvant for Mucosal Immunity to HIV-1 gp120 in Nonhuman Primates J. Immunol., December 1, 2004; 173(11): 6850 - 6857. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kataoka, J. R. McGhee, R. Kobayashi, K. Fujihashi, S. Shizukuishi, and K. Fujihashi Nasal Flt3 Ligand cDNA Elicits CD11c+CD8+ Dendritic Cells for Enhanced Mucosal Immunity J. Immunol., March 15, 2004; 172(6): 3612 - 3619. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. J. Berry, D. K. Hickey, K. A. Skelding, S. Bao, A. M. Rendina, P. M. Hansbro, C. M. Gockel, and K. W. Beagley Transcutaneous Immunization with Combined Cholera Toxin and CpG Adjuvant Protects against Chlamydia muridarum Genital Tract Infection Infect. Immun., February 1, 2004; 72(2): 1019 - 1028. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. N. Boyaka, A. Tafaro, R. Fischer, S. H. Leppla, K. Fujihashi, and J. R. McGhee Effective Mucosal Immunity to Anthrax: Neutralizing Antibodies and Th Cell Responses Following Nasal Immunization with Protective Antigen J. Immunol., June 1, 2003; 170(11): 5636 - 5643. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. N. Boyaka, M. Ohmura, K. Fujihashi, T. Koga, M. Yamamoto, M.-N. Kweon, Y. Takeda, R. J. Jackson, H. Kiyono, Y. Yuki, et al. Chimeras of Labile Toxin One and Cholera Toxin Retain Mucosal Adjuvanticity and Direct Th Cell Subsets Via Their B Subunit J. Immunol., January 1, 2003; 170(1): 454 - 462. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. P. Arulanandam, J. M. Lynch, D. E. Briles, S. Hollingshead, and D. W. Metzger Intranasal Vaccination with Pneumococcal Surface Protein A and Interleukin-12 Augments Antibody-Mediated Opsonization and Protective Immunity against Streptococcus pneumoniae Infection Infect. Immun., November 1, 2001; 69(11): 6718 - 6724. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kunisawa, T. Nakanishi, I. Takahashi, A. Okudaira, Y. Tsutsumi, K. Katayama, S. Nakagawa, H. Kiyono, and T. Mayumi Sendai Virus Fusion Protein-Mediates Simultaneous Induction of MHC Class I/II-Dependent Mucosal and Systemic Immune Responses Via the Nasopharyngeal-Associated Lymphoreticular Tissue Immune System J. Immunol., August 1, 2001; 167(3): 1406 - 1412. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhang, A. W. Masi, V. Barniak, K. Mountzouros, M. K. Hostetter, and B. A. Green Recombinant PhpA Protein, a Unique Histidine Motif-Containing Protein from Streptococcus pneumoniae, Protects Mice against Intranasal Pneumococcal Challenge Infect. Immun., June 1, 2001; 69(6): 3827 - 3836. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. W. van Ginkel, R. J. Jackson, Y. Yuki, and J. R. McGhee Cutting Edge: The Mucosal Adjuvant Cholera Toxin Redirects Vaccine Proteins into Olfactory Tissues J. Immunol., November 1, 2000; 165(9): 4778 - 4782. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Koga, J. R. McGhee, H. Kato, R. Kato, H. Kiyono, and K. Fujihashi Evidence For Early Aging in the Mucosal Immune System J. Immunol., November 1, 2000; 165(9): 5352 - 5359. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Agren, E. Sverremark, L. Ekman, K. Schon, B. Lowenadler, C. Fernandez, and N. Lycke The ADP-Ribosylating CTA1-DD Adjuvant Enhances T Cell-Dependent and Independent Responses by Direct Action on B Cells Involving Anti-Apoptotic Bcl-2- and Germinal Center-Promoting Effects J. Immunol., June 15, 2000; 164(12): 6276 - 6286. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kodama, S. Suenaga, T. Hirano, M. Suzuki, and G. Mogi Induction of Specific Immunoglobulin A and Th2 Immune Responses to P6 Outer Membrane Protein of Nontypeable Haemophilus influenzae in Middle Ear Mucosa by Intranasal Immunization Infect. Immun., April 1, 2000; 68(4): 2294 - 2300. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Briles, E. Ades, J. C. Paton, J. S. Sampson, G. M. Carlone, R. C. Huebner, A. Virolainen, E. Swiatlo, and S. K. Hollingshead Intranasal Immunization of Mice with a Mixture of the Pneumococcal Proteins PsaA and PspA Is Highly Protective against Nasopharyngeal Carriage of Streptococcus pneumoniae Infect. Immun., February 1, 2000; 68(2): 796 - 800. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. P. Simmons, P. Mastroeni, R. Fowler, M. Ghaem-maghami, N. Lycke, M. Pizza, R. Rappuoli, and G. Dougan MHC Class I-Restricted Cytotoxic Lymphocyte Responses Induced by Enterotoxin-Based Mucosal Adjuvants J. Immunol., December 15, 1999; 163(12): 6502 - 6510. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. W. Pascual, D. M. Hone, S. Hall, F. W. van Ginkel, M. Yamamoto, N. Walters, K. Fujihashi, R. J. Powell, S. Wu, J. L. Vancott, et al. Expression of Recombinant Enterotoxigenic Escherichia coli Colonization Factor Antigen I by Salmonella typhimurium Elicits a Biphasic T Helper Cell Response Infect. Immun., December 1, 1999; 67(12): 6249 - 6256. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Yamamoto, H. Kiyono, S. Yamamoto, E. Batanero, M.-N. Kweon, S. Otake, M. Azuma, Y. Takeda, and J. R. McGhee Direct Effects on Antigen-Presenting Cells and T Lymphocytes Explain the Adjuvanticity of a Nontoxic Cholera Toxin Mutant J. Immunol., June 15, 1999; 162(12): 7015 - 7021. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Yanagita, T. Hiroi, N. Kitagaki, S. Hamada, H.-o Ito, H. Shimauchi, S. Murakami, H. Okada, and H. Kiyono Nasopharyngeal-Associated Lymphoreticular Tissue (NALT) Immunity: Fimbriae-Specific Th1 and Th2 Cell-Regulated IgA Responses for the Inhibition of Bacterial Attachment to Epithelial Cells and Subsequent Inflammatory Cytokine Production J. Immunol., March 15, 1999; 162(6): 3559 - 3565. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 100 ng of Psp A plus 0.5 µg of nCT (
), or
PspA alone (
) on days 0, 7, and 14 and serum Ab titers were assessed
on day 21. Mononuclear cells were isolated from spleen, CLN, and lung
tissues and then examined by ELISPOT. The results are expressed as the
mean ± SEM obtained for five mice per group. The data are
representative of three separate experiments.
). The results are expressed as the mean
± SEM from five mice per group and were taken from a total of three
separate experiments. IL-4 was not detected in cells incubated with
rIL-2 alone at both mRNA and protein levels. The mRNA levels of IFN-
), 100 ng of PspA plus 0.5 µg of nCT (•), 100 ng of PspA
alone (
) on days 0, 7, and 14. Four weeks after the last immunization,
mice were challenged i.v. with 2.5 x 106 CFU of
capsular serotype 3 S. pneumoniae A66. PspA + mCT vs
nonimmunized group (
) at p = 0.0009; PspA + nCT
vs nonimmunized group at p = 0.0011.