In Vivo Activation of Naive CD4+ T Cells in Nasal Mucosa-Associated Lymphoid Tissue following Intranasal Immunization with Recombinant Strep
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感染与免疫杂志 2006年第5期
Laboratorio di Microbiologia Molecolare e Biotecnologia, Dipartimento di Biologia Molecolare, Universita di Siena, 53100 Siena, Italy
Department of Developmental and Surgical Sciences, University of Minnesota School of Dentistry, Minneapolis, Minnesota
ABSTRACT
The antigen-specific primary activation of CD4+ T cells was studied in vivo by adoptive transfer of ovalbumin-specific transgenic T cells (KJ1-26+ CD4+) following intranasal immunization with recombinant Streptococcus gordonii. A strain of S. gordonii expressing on its surface a model vaccine antigen fused to the ovalbumin (OVA) peptide from position 323 to 339 was constructed and used to study the OVA-specific T-cell activation in nasal mucosa-associated lymphoid tissue (NALT), lymph nodes, and spleens of mice immunized by the intranasal route. The recombinant strain, but not the wild type, activated the OVA-specific CD4+ T-cell population in the NALT (89% of KJ1-26+ CD4+ T cells) just 3 days following immunization. In the cervical lymph nodes and in the spleen, the percentage of proliferating cells was initially low, but it reached the peak of activation at day 5 (90%). This antigen-specific clonal expansion of KJ1-26+ CD4+ T cells after intranasal immunization was obtained with live and inactivated recombinant bacteria, and it indicates that the NALT is the site of antigen-specific T-cell priming.
INTRODUCTION
Intranasal immunization is very efficient at inducing humoral and cellular immune responses in the respiratory mucosa and at distant mucosal sites such as the genital tract and the gut (2, 12, 13, 16, 17, 51-53). It has been shown that intranasal immunization is more effective than oral and vaginal immunization at inducing generalized mucosal and systemic immune responses (7, 10, 27, 48, 50), and it requires smaller amounts of immunogens (51).
The nasal-associated lymphoid tissue (NALT) present in rodents is an important inductive tissue for the generation of mucosal immunity to inhaled antigens, capable of disseminating effector cells at distant mucosal sites (13, 48, 54). It is constituted of two bilateral strips of unencapsulated lymphoid cell aggregates and is considered functionally equivalent to the Waldeyer's ring present in humans (15, 45). The NALT is covered by an epithelium containing M cells, which closely resemble those present in the Peyer's patches (PP) of the gut and play an important role in antigen uptake for initiation of respiratory immune responses (13, 15, 35, 45). M cells present in the NALT may also represent an important site of entry of respiratory pathogens, such as group A Streptococcus (35). NALT has immunological characteristics and biological functions similar to those of PP and bronchus-associated lymphoid tissue (3, 9, 13). Unlike PP, the NALT develops after birth upon exposure to antigens and reaches its maximal size by 8 weeks of age (13). In unstimulated animals, NALT is a naive immunological site (49) characterized by unswitched immunoglobulin M-positive (IgM+) and IgD+ B cells and naive Th0 cells (11) that can differentiate into Th1 or Th2 cells, depending on the characteristics of the intranasally administered antigen (3, 13). An atypical B220low CD3low CD4– CD8– c-kit+ TLR2+ regulatory T cell has been recently observed in the NALT (40). NALT has been shown to also be an important site for generation of long-term immune memory (43, 48).
Effective nasal immunization requires an appropriate antigen formulation. Intranasal administration of pure protein antigens alone may not efficiently induce mucosal and systemic immune responses (27, 42, 50) and may instead elicit anergy and systemic tolerance (46, 47, 50). Different adjuvants and delivery systems have therefore been proposed for the development of effective nasal vaccines (13).
Our approach to intranasal immunization employs nonpathogenic gram-positive commensal bacteria as vaccine vectors (25, 30). We have developed a strategy for the expression of heterologous antigens on the surface of Streptococcus gordonii, a component of the normal microbial flora of the human oral cavity (32). A genetic system that allows the expression of two heterologous proteins on the surface of the same bacterial cell has also been developed (20). A variety of antigens, of different origins and sizes, have been expressed on the surface of S. gordonii (25, 30) and were shown to be immunogenic by the systemic and mucosal routes (oral, vaginal, and intragastric) in both mice and monkeys (6, 22-24, 26, 29, 31, 41). S. gordonii is efficiently internalized by both human and mouse dendritic cells (DCs), inducing their maturation and activation (4, 5, 39), and the heterologous antigen is presented by DCs in association with not only major histocompatibility complex (MHC) class II but also MHC class I molecules (39). Most importantly, it has recently been shown in a phase I clinical trial that S. gordonii is safe in humans when administered by the nasal/oral route and that it can be rapidly eradicated either spontaneously or with antibiotic treatment (14).
The aim of the present study was to assess whether intranasal immunization with recombinant S. gordonii is capable of priming naive antigen-specific CD4+ T lymphocytes at mucosal and systemic sites. Since the precursor frequency of naive antigen-specific CD4+ T cells is low, it is difficult to track their behavior. The adoptive transfer model system uses transgenic T lymphocytes with a T-cell receptor (TCR) that is specific for the H-2d-restricted Th epitope of ovalbumin from position 323 to 339 (OVA323-339) (33). Our recombinant model strain of S. gordonii expressing on the surface OVA323-339 in combination with the vaccine antigen P27 of simian immunodeficiency virus (SIV) was perfectly suited to study the in vivo induction of OVA-specific CD4+ T cells in NALT, draining lymph nodes, and spleen.
MATERIALS AND METHODS
Bacterial strains and growth conditions. S. gordonii GP1422, expressing the P27 protein of SIV, and the recipient control strain GP1295 (32) were used for immunization experiments. Bacteria were grown at 37°C with 5% CO2 in tryptic soy broth (TSB) (Difco, Detroit, MI), except for the cytofluorimetric analysis experiments, when they were cultured in TSB without dextrose (Difco). For solid medium, tryptic soy agar (Difco) supplemented with 2% horse blood was used. Antibiotics were added to the following concentrations: erythromycin, 1 μg/ml; kanamycin and streptomycin, 500 μg/ml. Inactivated bacteria were obtained by incubation with 0.9% formaldehyde solution (stock, 40% [wt/vol]; Farmitalia Carlo Erba) for 1 h at 25°C.
Construction of recombinant S. gordonii GP1422. An S. gordonii strain that surface displayed the P27 protein of SIV was constructed by using the host-vector system previously described (32). The emm6-based gene fusion includes (i) the DNA coding for OVA323-339 (44), (ii) the sequence coding for the P27 protein of SIV (accession no. M33262), and (iii) the E-tag-coding sequence. Construction of the above-mentioned emm6-based gene fusion required three cloning steps. Oligonucleotides OVA-F (5'-GGT ACC GGA TCC ATT TCT CAA GCT GTT CAT GCC GCT CAT GCA GAA ATT AAT GAA GCT GGT CGT GAT ATC-3'; KpnI, BamHI, and EcoRV restriction sites are underlined) and OVA-R (5'-GAT ATC ACG ACC AGC TTC-3'; an EcoRV restriction site is underlined) were used as a template to synthesize OVA by using the Klenow enzyme (Roche Applied Science, Monza, Italy); the fragment was cloned into restriction sites KpnI and EcoRV of plasmid pSMB55 (32), generating plasmid pSMB342. Cloning of the E-tag sequence into pSMB342 was achieved by ligating the SalI-PstI fragment of plasmid pSMB293 (M. R. Spinosa et al., unpublished data) into pSMB342 digested with the same enzymes. The resulting plasmid was named pSMB346. Finally, the P27-coding sequence was produced by PCR (with annealing at 58°C for 30 s, extension at 72°C for 75 s, and denaturation at 92°C for 20 s for a total of 30 cycles) by using primers Gag-F (5'-CCT AGG CCA GTA CAA CAA ATA GGT GGT AAC-3'; an AvrII restriction site is underlined) and Gag-R (5'-GTC GAC TGA ACC TCC AGA ACC GCC TGA TCC ACC AGA GCC ACC CTG GTC TCC TCC AAA GAG AG-3'; a SalI restriction site is underlined and a 36-bp sequence encoding a 12-amino-acid [aa] linker peptide is in italics) and plasmid SIV-X2 as the template. Plasmid SIV-X2, containing the genome of SIVmac239 (accession no. M33262) was kindly provided by K. Uberla, Bochum University, Germany. The PCR product was digested with AvrII and SalI and cloned into plasmid pSMB346 cut with XbaI and SalI. The resulting plasmid, designated pSMB450, was used to transform competent cells of S. gordonii GP1295. An erythromycin/streptomycin-resistant transformant that carried the expected emm6-based gene fusion was selected and designated GP1422. Procedures for cloning gene fusions in Escherichia coli, transformation of S. gordonii, and scoring and genetic analysis of streptococcal transformants were performed as previously described (37).
Analysis of recombinant bacteria. Western blotting was performed on the bacterial fraction containing surface proteins. The method for cell fractionation of S. gordonii has been previously described (38). Cell surface proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Protran; Schleicher and Schuell, Dassel, Germany). The presence of the M6/OVA/P27 fusion protein was assayed by using an M6-specific rabbit serum (1:1,000) and alkaline phosphatase-conjugated goat anti-rabbit antibody (1:10,000; Southern Biotechnology Associates, Birmingham, AL). For flow cytometric analysis, bacteria were grown in TSB without dextrose to early stationary phase and harvested, and approximately 108 CFU was used. Bacterial cells were washed, resuspended in phosphate-buffered saline (pH 7.4) (PBS) containing 1% bovine serum albumin, and incubated at 37°C for 30 min. Cells were incubated with anti-P27 rabbit polyclonal antibodies (1:1,000; Intracell, London, United Kingdom) or anti-E-tag antibody (Amersham Pharmacia) for 1 h at 4°C. After two washes with PBS, fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (1:64; Sigma-Aldrich, Munich, Germany) was added to bacteria and left for 20 min at 37°C. Cells were washed twice in PBS and finally resuspended in 0.5 ml of PBS and analyzed by flow cytometry (FACScan; Becton Dickinson, San Diego, CA).
Mice. Eight-week-old male DO11.10 TCR transgenic mice (H-2d) (28) and BALB/cJ mice were purchased from Jackson Laboratory (Maine) and maintained in the animal facilities at the University of Siena. Transgenic mice were maintained under specific-pathogen-free conditions. All animal procedures were in accordance with institutional guidelines.
Adoptive transfer of DO11.10 T cells. DO11.10 transgenic mice were sacrificed, and lymphoid organs (superficial and posterior cervical, brachial, axillary, mesenteric, and pelvic lymph nodes and spleen) were removed by dissection. Tissues were mashed onto a nylon screen (Sefar Italia, Italy), and the cells obtained were pooled, washed twice in Hanks' balanced salt solution (HBSS) (Sigma), and resuspended at 5 x 107/ml. The frequency of DO11.10 T cells was determined by labeling a cell aliquot with FITC-conjugated anti-CD3 (BD PharMingen, San Diego, CA), phycoerythrin (PE)-conjugated anti-KJ1-26 (Caltag Laboratories, Burlingame, CA), and Cy-Chrome-conjugated anti-CD4 (BD PharMingen) monoclonal antibodies (MAbs) and analyzed by using a FACscan flow cytometer. Pooled cells were then stained with carboxy-fluorescein diacetate succinimidyl ester (CFSE) (7.5 μM) for 10 min at 37°C and injected into the tail veins of recipient mice (1 x 106 to 2 x 106 cells/mice).
Intranasal immunization. Twenty-four hours after adoptive transfer of CFSE-labeled DO11.10 T cells, three groups of BALB/cJ mice were intranasally immunized with live and inactivated recombinant strain GP1422 or with the control strain GP1295. Mice were lightly anesthetized by intraperitoneal injection of tiletamine and zolazepam hydrochloride (Zoletil 20; Laboratoires Virbac, France) (6 mg/kg of body weight) and xylazine (Xilor 2%; Bio 98 Srl, Italy) (3 mg/kg) and then inoculated into a nostril with 109 CFU of bacteria in a total volume of 30 μl PBS. Three mice for each group were sacrificed at 3, 5, and 8 days following immunization.
Collection of NALT and lymphoid organs. NALT, cervical lymph nodes, and spleen were harvested from each mouse and immediately put in ice-cold Click's medium (Sigma-Aldrich). NALT samples were obtained as previously described (1, 34). Briefly, mice were euthanatized and decapitated. Their heads were immobilized, and the lower jaw and the tongue were removed. Then, using an 18-gauge needle (BM Microlance 3; BD Drogheda Ireland), palates were cut off by scoring along the outer edge distal to the central incisors and lateral to the hard palate through the oral mucosa, going distal up to the mesial surface of the first molar. With gentle lifting of the hard palate, the NALT remained attached to it. NALTs harvested from three mice were pooled. Collected organs were mashed onto a nylon screen and washed twice in HBSS prior to cell staining for flow cytometric analysis.
Flow cytometric analysis. Single-cell suspensions from NALT, cervical lymph nodes, and spleens were incubated with an Fc-blocking solution (0.5 mg CD16/CD32 MAb [clone 2.4G2; BD PharMingen], 5% mouse serum [Sigma-Aldrich], 5% rat serum [Sigma-Aldrich], 0.2% sodium azide [Acros Organics, New Jersey] in 100 ml of HBSS) for 30 min on ice. Cells were then stained with PE-conjugated anti-clonotypic KJ1-26 MAb (Caltag Laboratories) and CyChrome-conjugated anti-CD4 (BD PharMingen) (20 μl/106 cells) for 30 min at 4°C. Cells were then washed and resuspended in Click's medium and analyzed by flow cytometry. Sample acquisition stopped when 2,000 KJ1-26+ CD4+ events with light scatter properties of lymphocytes were acquired. Data were reported as the percentage of proliferating KJ1-26+ CD4+ T lymphocytes that have diluted CFSE in the CFSE (FL I) histogram. Data analysis was performed by using the CellQuest software (Becton Dickinson). The proliferative index (PI) was calculated as the ratio between the sum of the events in each generation and the calculated number of original parent cells (obtained by dividing the number of the events in each generation by 2 raised to the power of the generation number), as previously described (18).
RESULTS
Construction of recombinant S. gordonii strain GP1422. An S. gordonii strain displaying at the cell surface a fusion protein containing the model vaccine antigen P27 of SIV fused to the OVA323-239 peptide was constructed and named GP1422. The recombinant protein comprises OVA323-239 and P27 flanked by the first 122 NH2-terminal and the last 140 COOH-terminal amino acid residues of the M6 protein (Fig. 1A). The E-tag peptide was introduced into the fusion protein to allow flow cytometric analysis with antitag monoclonal antibody. In order to allow more flexibility to each protein/peptide, linkers were introduced between OVA323-339 and P27 and between P27 and the E-tag.
Surface display of the M6/P27 fusion protein by S. gordonii GP1422 was demonstrated by Western blot and flow cytometric analyses. Western blotting of streptococcal surface proteins extracted from GP1422 showed a protein band of approximately 90 kDa (the expected molecular mass is 81 kDa), while no reactivity was present in the GP1295 parent strain samples (Fig. 1B). The presence of the M6/P27 fusion protein at the surface of S. gordonii GP1422 was further assessed by flow cytometry on whole streptococcal cells by using anti-P27 polyclonal antibodies (Fig. 1C) and anti-E-tag MAb (data not shown). Ninety-eight percent of bacterial cells were positive for the expression of P27, with a mean fluorescence intensity of 58, while the control strain had a mean fluorescence value of 9 (Fig. 1C).
Recombinant S. gordonii GP1422 is therefore a vaccine vector for a SIV antigen, also harboring OVA323-339 as a model peptide to assess in vivo the primary activation of antigen-specific CD4+ T cells using the adoptive transfer model system.
KJ1-26+ CD4+ T-cell distribution in mice immunized with recombinant S. gordonii GP1422. In order to assess the distribution of DO11.10 T cells following adoptive transfer, we tested the presence of transgenic cells in lymphoid organs of recipient BALB/c mice intranasally immunized with recombinant S. gordonii GP1422 and with the control strain GP1295. DO11.10 T cells, expressing the transgenic TCR that recognizes chicken OVA323-339 in the context of I-Ad (44), can be easily tracked in vivo by using monoclonal antibodies specific for CD4 and the clonotypic TCR (KJ1-26) (8).
In mice immunized with the control strain GP1295, the number of KJ1-26+ CD4+ T cells was very low in lymph nodes and spleen and almost undetectable in the NALT (Fig. 2), as observed in untreated animals (data not shown). The percentage of KJ1-26+ CD4+ T cells increased in the three lymphoid organs following immunization with recombinant bacteria carrying the OVA323-339 peptide. A time course analysis of the presence of KJ1-26+ CD4+ T cells in the NALT showed the highest percentage of transgenic cells 3 and 5 days after immunization (Fig. 2). The amount of KJ1-26+ CD4+ T cells in lymph nodes and spleen was smaller at day 3 and peaked at day 5. Among the three secondary lymphoid organs analyzed, the lowest number of transgenic KJ1-26+ CD4+ T cells was observed in the spleen (Fig. 2).
These data demonstrate that the clonal expansion of KJ1-26+ CD4+ T cells is antigen specific and not due to other vector antigens.
Antigen-specific T-cell activation in lymphoid organs. To study proliferation of DO11.10 T cells in vivo, transgenic cells were labeled with CFSE before injection into syngeneic recipient BALB/c mice. CFSE is a vital dye that can label cells very stably by covalently coupling to intracellular molecules, and it can be used to monitor lymphocyte proliferation due to the progressive halving of the dye fluorescence following cell division (19).
Mice inoculated with the control strain GP1295 maintained a high level of CFSE fluorescence in the three organs, indicating that no cell division had occurred (Fig. 3). As mentioned above, in the NALT of control mice the number of KJ1-26+ CD4+ T cells was undetectable. OVA-specific T cells proliferated in the NALT, lymph nodes, and spleens of mice immunized with the GP1422 strain carrying the OVA peptide, as shown by the progressive dilution of CFSE in the three organs (Fig. 3).
The time course analysis of the KJ1-26+ CD4+ T-cell activation performed 3, 5, and 8 days following immunization showed a high level of proliferation in the NALT from the third day, with 80% of proliferating cells; this reached almost 90% at day 5 and was maintained at the same values up to day 8 (Fig. 4A). In the draining lymph nodes and mainly in the spleen, the percentages of proliferating cells were lower at day 3 (57% and 14%, respectively) (Fig. 4B and C); they reached the peak of activation of approximately 90% in both organs at day 5 and declined at day 8. At day 5 approximately 90% of KJ1-26+ CD4+ T cells had divided between one and eight times in mice immunized with live GP1422 strain (Fig. 3). Inactivated recombinant S. gordonii also was able to stimulate a clear OVA-specific clonal expansion of KJ1-26+ CD4+ T cells (about 80% of transgenic dividing cells), although a higher proportion of still-undivided cells was present both in the lymph nodes and spleen (Fig. 3). When the PI was calculated, similar values were obtained for the NALTs of mice immunized with live and inactivated GP1422 (5 and 4.2, respectively). The PI was instead 1.85-fold higher in the lymph nodes and spleens of mice immunized with live bacteria (4.5 and 5.5, respectively) than in those treated with inactivated bacteria (2.4 and 3, respectively). Mice treated with the control strain GP1295 did not respond at any time points in any lymphoid organs tested (Fig. 4).
The delayed appearance of KJ1-26+ CD4+ T cells with diluted CFSE in lymph nodes and spleen following immunization suggests either a migration of activated antigen-specific CD4+ T cells from NALT to other secondary lymphoid organs or a delayed appearance of antigen-presenting cells loaded with S. gordonii migrating from the nose to the draining cervical lymph nodes.
DISCUSSION
The model of adoptive transfer of transgenic T lymphocytes (33) was used to demonstrate that intranasal immunization with the recombinant commensal bacterium S. gordonii is effective at inducing primary activation of antigen-specific CD4+ T cells in vivo, which is essential for the development of long-term immunity to vaccine antigens.
S. gordonii has been previously used as a vaccine vector for expression of a variety of antigens (25, 30), and it was shown to be immunogenic by systemic and mucosal routes both in mice and monkeys (6, 22-24, 26, 29, 31, 41). Furthermore, S. gordonii administered by the nasal/oral route has been recently shown to be safe in a phase I trial in 150 human volunteers, and it can be rapidly eradicated either spontaneously or with antibiotic treatment (14), suggesting that the use of S. gordonii is feasible in humans.
In the present study, a strong proliferative response of OVA-specific T cells was observed in the NALT, draining lymph nodes, and spleens of mice immunized with recombinant S. gordonii expressing the model fusion protein containing OVA. The cell activation observed towards the OVA T-helper epitope could be representative of CD4+ T-cell responses also induced towards T-helper epitopes of the model fusion protein. The highest levels of proliferation were observed 5 days following intranasal immunization, when about 90% of KJ1-26+ CD4+ T cells had divided between one and eight times. This is in accordance with what was observed with the human pathogen Streptococcus pyogenes administered by the nasal route when a comparable dose of inoculum was used (34). The antigen-specific CD4+ T-cell response was observed first in the NALT, already at 3 days following immunization, as previously shown with group A Streptococcus (34). This behavior is also similar to that observed in the gut following oral infection with Salmonella, which induces a T-cell response mainly in PP (21).
It is important to highlight that inactivated recombinant S. gordonii also was effective at inducing T-cell activation in the NALT and in the other secondary lymphoid organs analyzed; this is in agreement with recent data of ours indicating that inactivated recombinant S. gordonii expressing a meningococcal vaccine antigen is capable of inducing antigen-specific humoral responses following repeated intranasal immunizations (A. Ciabattini et al., unpublished data). These results open the potential of using inactivated recombinant commensal bacteria for intranasal immunization, thereby further circumventing the safety issues related to the use of live vaccine vectors.
Interestingly, in mice immunized with the wild-type strain, the number of OVA-specific CD4+ T cells (KJ1-26+) following adoptive transfer was low in lymph nodes and spleen and undetectable in the NALT, while it was increased in the three lymphoid organs following immunization with recombinant bacteria carrying the OVA323-339 peptide. This observation indicates that the KJ1-26+ CD4+ T-cell expansion is OVA specific and not due to vector antigens.
We have previously shown in vitro that human and mouse DCs process and present the vaccine antigens delivered by S. gordonii to CD4+ T cells much more efficiently than the soluble form (4, 39), and they also are more potent than B cells at presenting an MHC class II-restricted antigen expressed on the bacterial surface (5). As DCs have repeatedly been shown to be crucial for activation of naive T cells, we can therefore hypothesize that DCs present in the nasal mucosa (3, 9, 36) might be stimulated in vivo by recombinant S. gordonii, thereby inducing the efficient T-cell activation observed.
Repeated intranasal immunizations with recombinant S. gordonii were already shown to be effective at inducing local and systemic antibody responses capable of conferring protection (22). By using the adoptive transfer model system, we have demonstrated for the first time the efficient intranasal priming of naive antigen-specific CD4+ T cells against antigens expressed by live and inactivated recombinant S. gordonii in NALT, draining lymph nodes, and spleen. Taken together, these data further support the use of S. gordonii as an effective mucosal vaccine vector capable of inducing humoral and cellular immunity, both locally and systemically.
ACKNOWLEDGMENTS
We thank Riccardo Parigi for his skillful help with the animals.
This study was carried out with financial support from the Commission of the European Communities, Sixth Framework Programme, contract LSHP-CT-2003-503240, Mucosal Vaccines for Poverty-Related Diseases (MUVAPRED), and Fifth Framework Programme, contract QLK2-CT-2002-00882, Mucosal Vaccines Against Human and Simian Immunodeficiency Viruses Based on Dendritic Cells (MUVADEN) (to G.P.); from "Azione Concertata Italiana per lo Sviluppo di un Vaccino HIV/AIDS" (ICAV) of the Istituto Superiore di Sanita contract no. 45F.29 and MIUR (FIRB RBNE01RB9B_009) (to D.M.); and from the University of Minnesota School of Dentistry New Faculty Recruitment Fund (to M.C.).
REFERENCES
1. Asanuma, H., A. H. Thompson, T. Iwasaki, Y. Sato, Y. Inaba, C. Aizawa, T. Kurata, and S. Tamura. 1997. Isolation and characterization of mouse nasal-associated lymphoid tissue. J. Immunol. Methods 202:123-131.
2. Bergquist, C., E. L. Johansson, T. Lagergard, J. Holmgreen, and A. Rudin. 1997. Intranasal vaccination of humans with recombinant cholera toxin B subunit induces systemic and local antibody responses in the upper respiratory tract and the vagina. Infect. Immun. 65:2676-2684.
3. Bienenstock, J., and M. R. McDermott. 2005. Bronchus- and nasal-associated lymphoid tissue. Immunol. Rev. 206:22-31.
4. Corinti, S., D. Medaglini, A. Cavani, M. Rescigno, G. Pozzi, P. Ricciardi-Castagnoli, and G. Girolomoni. 1999. Human dendritic cells very efficiently present a heterologous antigen expressed on the surface of recombinant Gram-positive bacteria to CD4+ T lymphocytes. J. Immunol. 163:3029-3036.
5. Corinti, S., D. Medaglini, C. Prezzi, G. Pozzi, and G. Girolomoni. 2000. Human dendritic cells are superior to B cells at presenting a major histocompatibility complex class-II restricted heterologous antigen expressed on recombinant Streptococcus gordonii. Infect. Immun. 68:1879-1883.
6. Di Fabio, S., D. Medaglini, C. M. Rush, G. Corrias, G. L. Panzini, M. Pace, P. Verani, G. Pozzi, and F. Titti. 1998. Vaginal immunization of Cynomolgus monkeys with Streptococcus gordonii expressing HIV-1 and HPV 16 antigens. Vaccine 16:485-492.
7. Di Tommaso, A., G. Saletti, M. G. Pizza, R. Rappuoli, and G. Dougan. 1996. Induction of antigen-specific antibodies in vaginal secretions by using a nontoxic mutant of heat-labile enterotoxin as a mucosal adjuvant. Infect. Immun. 64:974-979.
8. Haskins, K., R. Kubo, J. White, M. Pigeon, J. Kappler, and P. Marrack. 1983. The major histocompatibility complex-restricted antigen receptor on T cells. I. Isolation with a monoclonal antibody. J. Exp. Med. 157:1149-1169.
9. Heritage, P. L., B. J. Underdown, A. L. Arsenault, D. P. Snider, and M. R. McDermott. 1997. Comparison of murine nasal-associated lymphoid tissue and Peyer's patches. Am. J. Respir. Crit. Care Med. 156:1256-1262.
10. Hirabayashi, Y., H. Kurata, H. Funato, T. Nagamine, C. Aizawa, S. Tamura, K. Shimada, and T. Kurata. 1990. Comparison of intranasal inoculation of influenza HA vaccine combined with cholera toxin B subunit with oral or parenteral vaccination. Vaccine 8:243-248.
11. Hiroi, T., K. Iwatani, H. Iijima, S. Kodama, M. Yanagita, and H. Kiyono. 1998. Nasal immune system: distinctive Th0 and Th1/Th2 type environments in murine nasal-associated lymphoid tissues and nasal passage, respectively. Eur. J. Immunol. 28:3346-3353.
12. Imaoka, K., C. J. Miller, M. Kubota, M. B. McChesney, B. Lohman, M. Yamamoto, K. Fujihashi, K. Someya, M. Honda, J. R. McGhee, and H. Kiyono. 1998. Nasal immunization of nonhuman primates with simian immunodeficiency virus p55gag and cholera toxin adjuvant induces Th1/Th2 help for virus-specific immune responses in reproductive tissues. J. Immunol. 161:5952-5958.
13. Kiyono, H., and S. Fukuyama. 2004. NALT- versus Peyer's-patch-mediated mucosal immunity. Nat. Rev. Immunol. 4:699-710.
14. Kotloff, K. L., S. S. Wassermann, K. F. Jones, S. Livio, D. E. Hruby, C. A. Franke, and V. A. Fischetti. 2005. Clinical and microbiological responses of volunteers to combined intranasal and oral inoculation with a Streptococcus gordonii carrier strain intended for future use as a group A Streptococcus vaccine. Infect. Immun. 73:2360-2366.
15. Kuper, C. F., P. J. Koornstra, D. M. H. Hameleers, J. Biewenga, B. J. Spit, A. M. Duijevestijn, P. J. C. van Breda Vriesman, and T. Sminia. 1992. The role of nasopharyngeal lymphoid tissue. Immunol. Today 13:219-223.
16. Kurono, Y., M. Yamamoto, K. Fujihashi, S. Kodama, M. Suzuki, G. Mogi, J. R. McGhee, and H. Kiyono. 1999. Nasal immunization induces Haemophilus influenzae-specific Th1 and Th2 responses with mucosal IgA and systemic IgG antibodies for protective immunity. J. Infect. Dis. 180:122-132.
17. Langermann, S., S. Palaszynski, A. Sadziene, C. K. Stover, and S. Koenig. 1994. Systemic and mucosal immunity induced by BCG vector expressing outer-surface protein A of Borrelia burgdorferi. Nature 372:552-555.
18. Lyons, A. B. 2000. Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution. J. Immunol. Methods 243:147-154.
19. Lyons, A. B., and C. R. Parish. 1994. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171:131-137.
20. Maggi, T., M. R. Spinosa, S. Ricci, D. Medaglini, G. Pozzi, and M. R. Oggioni. 2002. Genetic engineering of Streptococcus gordonii for the simultaneous display of two heterologous proteins at the bacterial surface. FEMS Microbiol. Lett. 210:135-141.
21. McSorley, S. J., S. Asch, M. Costalonga, R. L. Reinhardt, and M. K. Jenkins. 2002. Tracking Salmonella-specific CD4 T cells in vivo reveals a local mucosal response to a disseminated infection. Immunity 16:365-377.
22. Medaglini, D., A. Ciabattini, M. R. Spinosa, T. Maggi, H. Marcotte, M. R. Oggioni, and G. Pozzi. 2001. Immunization with recombinant Streptococcus gordonii expressing tetanus toxin fragment C confers protection from lethal challenge in mice. Vaccine 19:1931-1939.
23. Medaglini, D., M. R. Oggioni, and G. Pozzi. 1998. Vaginal immunization with recombinant Gram positive bacteria. Am. J. Reprod. Immunol. 39:199-208.
24. Medaglini, D., G. Pozzi, T. P. King, and V. A. Fischetti. 1995. Mucosal and systemic immune responses to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii after oral colonization. Proc. Natl. Acad. Sci. USA 92:6868-6872.
25. Medaglini, D., S. Ricci, T. Maggi, C. M. Rush, R. Manganelli, M. R. Oggioni, and G. Pozzi. 1997. Recombinant Gram-positive bacteria as vehicles of vaccine antigens. Biotechnol. Annu. Rev. 3:297-312.
26. Medaglini, D., C. M. Rush, P. Sestini, and G. Pozzi. 1997. Commensal bacteria as vectors for mucosal vaccines against sexually transmitted diseases: vaginal colonization with recombinant streptococci induces local and systemic antibodies in mice. Vaccine 15:1330-1337.
27. Muller, C. P., P. Beauverger, F. Schneider, F. Jung, and N. H. Brons. 1995. Cholera toxin B stimulates systemic neutralizing antibodies after intranasal co-immunization with measles virus. J. Gen. Virol. 76:1371-1380.
28. Murphy, K. M., A. M. Heimberger, and D. Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+ CD8+ TCRlo thymocytes in vivo. Science 250:1720-1723.
29. Oggioni, M. R., R. Manganelli, M. Contorni, M. Tommasino, and G. Pozzi. 1995. Immunization of mice by oral colonization with live recombinant commensal streptococci. Vaccine 13:775-779.
30. Oggioni, M. R., D. Medaglini, T. Maggi, and G. Pozzi. 1999. Engineering the Gram-positive cell surface for construction of bacterial vaccine vectors. Methods 19:163-173.
31. Oggioni, M. R., D. Medaglini, L. Romano, F. Peruzzi, T. Maggi, L. Lozzi, L. Bracci, M. Zazzi, F. Manca, P. E. Valensin, and G. Pozzi. 1999. Antigenicity and immunogenicity of the V3 domain of HIV-1 gp120 expressed on the surface of Streptococcus gordonii. AIDS Res. Hum. Retroviruses 15:451-459.
32. Oggioni, M. R., and G. Pozzi. 1996. A host-vector system for heterologous gene expression in Streptococcus gordonii. Gene 169:85-90.
33. Pape, K. A., E. R. Kearney, A. Khoruts, A. Mondino, R. Merica, Z. M. Chen, E. Ingulli, J. White, J. G. Johnson, and M. K. Jenkins. 1997. Use of adoptive transfer of T-cell-antigen-receptor-transgenic T cell for the study of T-cell activation in vivo. Immunol. Rev. 156:67-78.
34. Park, H. S., M. Costalonga, R. L. Reinhardt, P. E. Dombek, M. K. Jenkins, and P. P. Cleary. 2004. Primary induction of CD4 T cell responses in nasal associated lymphoid tissue during group A streptococcal infection. Eur. J. Immunol. 34:2843-2853.
35. Park, H. S., K. P. Francis, J. Yu, and P. P. Cleary. 2003. Membranous cells in nasal-associated lymphoid tissue: a portal of entry for the respiratory mucosal pathogen group A Streptococcus. J. Immunol. 171:2532-2537.
36. Porgador, A., H. F. Staats, Y. Itoh, and B. L. Kelsall. 1998. Intranasal immunization with cytotoxic T-lymphocyte epitope peptide and mucosal adjuvant cholera toxin: selective augmentation of peptide-presenting dendritic cells in nasal mucosa-associated lymphoid tissue. Infect. Immun. 66:5876-5881.
37. Pozzi, G., R. A. Musmanno, P. M. J. Lievens, M. R. Oggioni, P. Plevani, and R. Manganelli. 1990. Methods and parameters for genetic transformation of Streptococcus sanguis Challis. Res. Microbiol. 141:659-670.
38. Pozzi, G., M. R. Oggioni, R. Manganelli, D. Medaglini, V. A. Fischetti, D. Fenoglio, M. T. Valle, A. Kunkl, and F. Manca. 1994. Human T-helper cell recognition of an immunodominant epitope of HIV-1 gp120 expressed on the surface of Streptococcus gordonii. Vaccine 12:1071-1077.
39. Rescigno, M., S. Citterio, C. Thery, M. Rittig, D. Medaglini, G. Pozzi, S. Amigorena, and P. Ricciardi-Castagnoli. 1998. Bacterial-induced neo-biosynthesis, stabilization and surface expression of functional class I molecules in mouse dendritic cells. Proc. Natl. Acad. Sci. USA 95:5229-5234.
40. Rharbaoui, F., D. Bruder, M. Vidakovic, T. Ebensen, J. Buer, and C. A. Guzman. 2005. Characterization of a B220+ lymphoid cell subpopulation with immune modulatory functions in nasal-associated lymphoid tissues. J. Immunol. 174:1317-1324.
41. Ricci, S., D. Medaglini, C. M. Rush, A. Marcello, R. Manganelli, G. Palù, and G. Pozzi. 2000. Immunogenicity of the B monomer of the Escherichia coli heat-labile toxin expressed on the surface of Streptococcus gordonii. Infect. Immun. 68:760-766.
42. Shahin, R., M. Leef, J. Eldridge, M. Hudson, and R. Gilley. 1995. Adjuvanticity and protective immunity elicited by Bordetella pertussis antigens encapsulated in poly(DL-lactide-co-glycolide) microspheres. Infect. Immun. 63:1195-1200.
43. Shimoda, M., T. Nakamura, Y. Takahashi, H. Asanuma, S. Tamura, T. Kurata, T. Mizuochi, N. Azuma, C. Kanno, and T. Takemori. 2001. Isotype-specific selection of high affinity memory B cells in nasal-associated lymphoid tissue. J. Exp. Med. 194:1597-1607.
44. Shimonkevitz, R., S. Colon, J. W. Kappler, P. Marrack, and H. M. Grey. 1984. Antigen recognition by H-2-restricted T cells. II. A tryptic ovalbumin peptide that substitutes for processed antigen. J. Immunol. 133:2067-2074.
45. Spit, B. J., E. G. Hendriksen, J. P. Bruijntjes, and C. F. Kuper. 1989. Nasal lymphoid tissue in the rat. Cell Tissue Res. 255:193-198.
46. Unger, W. W., W. Jansen, D. A. Wolvers, A. G. van Halteren, G. Kraal, and J. N. Samsom. 2003. Nasal tolerance induces antigen-specific CD4+CD25– regulatory T cells that can transfer their regulatory capacity to naive CD4+ T cells. Int. Immunol. 15:731-739.
47. Waldo, F. B., A. W. van den Wall Bake, J. Mestecky, and S. Husby. 1994. Suppression of the immune response by nasal immunization. Clin. Immunol. Immunopathol. 72:30-34.
48. Wu, H. Y., H. H. Nguyen, and M. W. Russel. 1997. Nasal lymphoid tissue (NALT) as a mucosal immune inductive site. Scand. J. Immunol. 46:506-513.
49. Wu, H. Y., E. B. Nikolova, K. W. Beagley, and M. W. Russell. 1996. Induction of antibody-secreting cells and T-helper and memory cells in murine nasal lymphoid tissue. Immunology 88:493-500.
50. Wu, H. Y., and M. W. Russel. 1997. Nasal lymphoid tissue, intranasal immunization, and compartmentalization of the common mucosal immune system. Immunol. Res. 16:187-201.
51. Wu, H. Y., and M. W. Russel. 1993. Induction of mucosal immunity by intranasal application of a streptococcal surface protein antigen with the cholera toxin B subunit. Infect. Immun. 61:314-322.
52. Yanagita, M., T. Hiroi, N. Kitagaki, S. Hamada, H. O. Ito, H. Shimauchi, S. Murakami, H. Okada, and H. Kiyono. 1999. 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. 162:3559-3565.
53. Zuercher, A. W. 2003. Upper respiratory tract immunity. Viral Immunol. 16:279-289.
54. Zuercher, A. W., S. E. Coffin, M. C. Thurnheer, P. Fundova, and J. J. Cebra. 2002. Nasal-associated lymphoid tissue is a mucosal inductive site for virus-specific humoral and cellular immune responses. J. Immunol. 168:1796-1803.(Donata Medaglini, Annalis)
Department of Developmental and Surgical Sciences, University of Minnesota School of Dentistry, Minneapolis, Minnesota
ABSTRACT
The antigen-specific primary activation of CD4+ T cells was studied in vivo by adoptive transfer of ovalbumin-specific transgenic T cells (KJ1-26+ CD4+) following intranasal immunization with recombinant Streptococcus gordonii. A strain of S. gordonii expressing on its surface a model vaccine antigen fused to the ovalbumin (OVA) peptide from position 323 to 339 was constructed and used to study the OVA-specific T-cell activation in nasal mucosa-associated lymphoid tissue (NALT), lymph nodes, and spleens of mice immunized by the intranasal route. The recombinant strain, but not the wild type, activated the OVA-specific CD4+ T-cell population in the NALT (89% of KJ1-26+ CD4+ T cells) just 3 days following immunization. In the cervical lymph nodes and in the spleen, the percentage of proliferating cells was initially low, but it reached the peak of activation at day 5 (90%). This antigen-specific clonal expansion of KJ1-26+ CD4+ T cells after intranasal immunization was obtained with live and inactivated recombinant bacteria, and it indicates that the NALT is the site of antigen-specific T-cell priming.
INTRODUCTION
Intranasal immunization is very efficient at inducing humoral and cellular immune responses in the respiratory mucosa and at distant mucosal sites such as the genital tract and the gut (2, 12, 13, 16, 17, 51-53). It has been shown that intranasal immunization is more effective than oral and vaginal immunization at inducing generalized mucosal and systemic immune responses (7, 10, 27, 48, 50), and it requires smaller amounts of immunogens (51).
The nasal-associated lymphoid tissue (NALT) present in rodents is an important inductive tissue for the generation of mucosal immunity to inhaled antigens, capable of disseminating effector cells at distant mucosal sites (13, 48, 54). It is constituted of two bilateral strips of unencapsulated lymphoid cell aggregates and is considered functionally equivalent to the Waldeyer's ring present in humans (15, 45). The NALT is covered by an epithelium containing M cells, which closely resemble those present in the Peyer's patches (PP) of the gut and play an important role in antigen uptake for initiation of respiratory immune responses (13, 15, 35, 45). M cells present in the NALT may also represent an important site of entry of respiratory pathogens, such as group A Streptococcus (35). NALT has immunological characteristics and biological functions similar to those of PP and bronchus-associated lymphoid tissue (3, 9, 13). Unlike PP, the NALT develops after birth upon exposure to antigens and reaches its maximal size by 8 weeks of age (13). In unstimulated animals, NALT is a naive immunological site (49) characterized by unswitched immunoglobulin M-positive (IgM+) and IgD+ B cells and naive Th0 cells (11) that can differentiate into Th1 or Th2 cells, depending on the characteristics of the intranasally administered antigen (3, 13). An atypical B220low CD3low CD4– CD8– c-kit+ TLR2+ regulatory T cell has been recently observed in the NALT (40). NALT has been shown to also be an important site for generation of long-term immune memory (43, 48).
Effective nasal immunization requires an appropriate antigen formulation. Intranasal administration of pure protein antigens alone may not efficiently induce mucosal and systemic immune responses (27, 42, 50) and may instead elicit anergy and systemic tolerance (46, 47, 50). Different adjuvants and delivery systems have therefore been proposed for the development of effective nasal vaccines (13).
Our approach to intranasal immunization employs nonpathogenic gram-positive commensal bacteria as vaccine vectors (25, 30). We have developed a strategy for the expression of heterologous antigens on the surface of Streptococcus gordonii, a component of the normal microbial flora of the human oral cavity (32). A genetic system that allows the expression of two heterologous proteins on the surface of the same bacterial cell has also been developed (20). A variety of antigens, of different origins and sizes, have been expressed on the surface of S. gordonii (25, 30) and were shown to be immunogenic by the systemic and mucosal routes (oral, vaginal, and intragastric) in both mice and monkeys (6, 22-24, 26, 29, 31, 41). S. gordonii is efficiently internalized by both human and mouse dendritic cells (DCs), inducing their maturation and activation (4, 5, 39), and the heterologous antigen is presented by DCs in association with not only major histocompatibility complex (MHC) class II but also MHC class I molecules (39). Most importantly, it has recently been shown in a phase I clinical trial that S. gordonii is safe in humans when administered by the nasal/oral route and that it can be rapidly eradicated either spontaneously or with antibiotic treatment (14).
The aim of the present study was to assess whether intranasal immunization with recombinant S. gordonii is capable of priming naive antigen-specific CD4+ T lymphocytes at mucosal and systemic sites. Since the precursor frequency of naive antigen-specific CD4+ T cells is low, it is difficult to track their behavior. The adoptive transfer model system uses transgenic T lymphocytes with a T-cell receptor (TCR) that is specific for the H-2d-restricted Th epitope of ovalbumin from position 323 to 339 (OVA323-339) (33). Our recombinant model strain of S. gordonii expressing on the surface OVA323-339 in combination with the vaccine antigen P27 of simian immunodeficiency virus (SIV) was perfectly suited to study the in vivo induction of OVA-specific CD4+ T cells in NALT, draining lymph nodes, and spleen.
MATERIALS AND METHODS
Bacterial strains and growth conditions. S. gordonii GP1422, expressing the P27 protein of SIV, and the recipient control strain GP1295 (32) were used for immunization experiments. Bacteria were grown at 37°C with 5% CO2 in tryptic soy broth (TSB) (Difco, Detroit, MI), except for the cytofluorimetric analysis experiments, when they were cultured in TSB without dextrose (Difco). For solid medium, tryptic soy agar (Difco) supplemented with 2% horse blood was used. Antibiotics were added to the following concentrations: erythromycin, 1 μg/ml; kanamycin and streptomycin, 500 μg/ml. Inactivated bacteria were obtained by incubation with 0.9% formaldehyde solution (stock, 40% [wt/vol]; Farmitalia Carlo Erba) for 1 h at 25°C.
Construction of recombinant S. gordonii GP1422. An S. gordonii strain that surface displayed the P27 protein of SIV was constructed by using the host-vector system previously described (32). The emm6-based gene fusion includes (i) the DNA coding for OVA323-339 (44), (ii) the sequence coding for the P27 protein of SIV (accession no. M33262), and (iii) the E-tag-coding sequence. Construction of the above-mentioned emm6-based gene fusion required three cloning steps. Oligonucleotides OVA-F (5'-GGT ACC GGA TCC ATT TCT CAA GCT GTT CAT GCC GCT CAT GCA GAA ATT AAT GAA GCT GGT CGT GAT ATC-3'; KpnI, BamHI, and EcoRV restriction sites are underlined) and OVA-R (5'-GAT ATC ACG ACC AGC TTC-3'; an EcoRV restriction site is underlined) were used as a template to synthesize OVA by using the Klenow enzyme (Roche Applied Science, Monza, Italy); the fragment was cloned into restriction sites KpnI and EcoRV of plasmid pSMB55 (32), generating plasmid pSMB342. Cloning of the E-tag sequence into pSMB342 was achieved by ligating the SalI-PstI fragment of plasmid pSMB293 (M. R. Spinosa et al., unpublished data) into pSMB342 digested with the same enzymes. The resulting plasmid was named pSMB346. Finally, the P27-coding sequence was produced by PCR (with annealing at 58°C for 30 s, extension at 72°C for 75 s, and denaturation at 92°C for 20 s for a total of 30 cycles) by using primers Gag-F (5'-CCT AGG CCA GTA CAA CAA ATA GGT GGT AAC-3'; an AvrII restriction site is underlined) and Gag-R (5'-GTC GAC TGA ACC TCC AGA ACC GCC TGA TCC ACC AGA GCC ACC CTG GTC TCC TCC AAA GAG AG-3'; a SalI restriction site is underlined and a 36-bp sequence encoding a 12-amino-acid [aa] linker peptide is in italics) and plasmid SIV-X2 as the template. Plasmid SIV-X2, containing the genome of SIVmac239 (accession no. M33262) was kindly provided by K. Uberla, Bochum University, Germany. The PCR product was digested with AvrII and SalI and cloned into plasmid pSMB346 cut with XbaI and SalI. The resulting plasmid, designated pSMB450, was used to transform competent cells of S. gordonii GP1295. An erythromycin/streptomycin-resistant transformant that carried the expected emm6-based gene fusion was selected and designated GP1422. Procedures for cloning gene fusions in Escherichia coli, transformation of S. gordonii, and scoring and genetic analysis of streptococcal transformants were performed as previously described (37).
Analysis of recombinant bacteria. Western blotting was performed on the bacterial fraction containing surface proteins. The method for cell fractionation of S. gordonii has been previously described (38). Cell surface proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Protran; Schleicher and Schuell, Dassel, Germany). The presence of the M6/OVA/P27 fusion protein was assayed by using an M6-specific rabbit serum (1:1,000) and alkaline phosphatase-conjugated goat anti-rabbit antibody (1:10,000; Southern Biotechnology Associates, Birmingham, AL). For flow cytometric analysis, bacteria were grown in TSB without dextrose to early stationary phase and harvested, and approximately 108 CFU was used. Bacterial cells were washed, resuspended in phosphate-buffered saline (pH 7.4) (PBS) containing 1% bovine serum albumin, and incubated at 37°C for 30 min. Cells were incubated with anti-P27 rabbit polyclonal antibodies (1:1,000; Intracell, London, United Kingdom) or anti-E-tag antibody (Amersham Pharmacia) for 1 h at 4°C. After two washes with PBS, fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (1:64; Sigma-Aldrich, Munich, Germany) was added to bacteria and left for 20 min at 37°C. Cells were washed twice in PBS and finally resuspended in 0.5 ml of PBS and analyzed by flow cytometry (FACScan; Becton Dickinson, San Diego, CA).
Mice. Eight-week-old male DO11.10 TCR transgenic mice (H-2d) (28) and BALB/cJ mice were purchased from Jackson Laboratory (Maine) and maintained in the animal facilities at the University of Siena. Transgenic mice were maintained under specific-pathogen-free conditions. All animal procedures were in accordance with institutional guidelines.
Adoptive transfer of DO11.10 T cells. DO11.10 transgenic mice were sacrificed, and lymphoid organs (superficial and posterior cervical, brachial, axillary, mesenteric, and pelvic lymph nodes and spleen) were removed by dissection. Tissues were mashed onto a nylon screen (Sefar Italia, Italy), and the cells obtained were pooled, washed twice in Hanks' balanced salt solution (HBSS) (Sigma), and resuspended at 5 x 107/ml. The frequency of DO11.10 T cells was determined by labeling a cell aliquot with FITC-conjugated anti-CD3 (BD PharMingen, San Diego, CA), phycoerythrin (PE)-conjugated anti-KJ1-26 (Caltag Laboratories, Burlingame, CA), and Cy-Chrome-conjugated anti-CD4 (BD PharMingen) monoclonal antibodies (MAbs) and analyzed by using a FACscan flow cytometer. Pooled cells were then stained with carboxy-fluorescein diacetate succinimidyl ester (CFSE) (7.5 μM) for 10 min at 37°C and injected into the tail veins of recipient mice (1 x 106 to 2 x 106 cells/mice).
Intranasal immunization. Twenty-four hours after adoptive transfer of CFSE-labeled DO11.10 T cells, three groups of BALB/cJ mice were intranasally immunized with live and inactivated recombinant strain GP1422 or with the control strain GP1295. Mice were lightly anesthetized by intraperitoneal injection of tiletamine and zolazepam hydrochloride (Zoletil 20; Laboratoires Virbac, France) (6 mg/kg of body weight) and xylazine (Xilor 2%; Bio 98 Srl, Italy) (3 mg/kg) and then inoculated into a nostril with 109 CFU of bacteria in a total volume of 30 μl PBS. Three mice for each group were sacrificed at 3, 5, and 8 days following immunization.
Collection of NALT and lymphoid organs. NALT, cervical lymph nodes, and spleen were harvested from each mouse and immediately put in ice-cold Click's medium (Sigma-Aldrich). NALT samples were obtained as previously described (1, 34). Briefly, mice were euthanatized and decapitated. Their heads were immobilized, and the lower jaw and the tongue were removed. Then, using an 18-gauge needle (BM Microlance 3; BD Drogheda Ireland), palates were cut off by scoring along the outer edge distal to the central incisors and lateral to the hard palate through the oral mucosa, going distal up to the mesial surface of the first molar. With gentle lifting of the hard palate, the NALT remained attached to it. NALTs harvested from three mice were pooled. Collected organs were mashed onto a nylon screen and washed twice in HBSS prior to cell staining for flow cytometric analysis.
Flow cytometric analysis. Single-cell suspensions from NALT, cervical lymph nodes, and spleens were incubated with an Fc-blocking solution (0.5 mg CD16/CD32 MAb [clone 2.4G2; BD PharMingen], 5% mouse serum [Sigma-Aldrich], 5% rat serum [Sigma-Aldrich], 0.2% sodium azide [Acros Organics, New Jersey] in 100 ml of HBSS) for 30 min on ice. Cells were then stained with PE-conjugated anti-clonotypic KJ1-26 MAb (Caltag Laboratories) and CyChrome-conjugated anti-CD4 (BD PharMingen) (20 μl/106 cells) for 30 min at 4°C. Cells were then washed and resuspended in Click's medium and analyzed by flow cytometry. Sample acquisition stopped when 2,000 KJ1-26+ CD4+ events with light scatter properties of lymphocytes were acquired. Data were reported as the percentage of proliferating KJ1-26+ CD4+ T lymphocytes that have diluted CFSE in the CFSE (FL I) histogram. Data analysis was performed by using the CellQuest software (Becton Dickinson). The proliferative index (PI) was calculated as the ratio between the sum of the events in each generation and the calculated number of original parent cells (obtained by dividing the number of the events in each generation by 2 raised to the power of the generation number), as previously described (18).
RESULTS
Construction of recombinant S. gordonii strain GP1422. An S. gordonii strain displaying at the cell surface a fusion protein containing the model vaccine antigen P27 of SIV fused to the OVA323-239 peptide was constructed and named GP1422. The recombinant protein comprises OVA323-239 and P27 flanked by the first 122 NH2-terminal and the last 140 COOH-terminal amino acid residues of the M6 protein (Fig. 1A). The E-tag peptide was introduced into the fusion protein to allow flow cytometric analysis with antitag monoclonal antibody. In order to allow more flexibility to each protein/peptide, linkers were introduced between OVA323-339 and P27 and between P27 and the E-tag.
Surface display of the M6/P27 fusion protein by S. gordonii GP1422 was demonstrated by Western blot and flow cytometric analyses. Western blotting of streptococcal surface proteins extracted from GP1422 showed a protein band of approximately 90 kDa (the expected molecular mass is 81 kDa), while no reactivity was present in the GP1295 parent strain samples (Fig. 1B). The presence of the M6/P27 fusion protein at the surface of S. gordonii GP1422 was further assessed by flow cytometry on whole streptococcal cells by using anti-P27 polyclonal antibodies (Fig. 1C) and anti-E-tag MAb (data not shown). Ninety-eight percent of bacterial cells were positive for the expression of P27, with a mean fluorescence intensity of 58, while the control strain had a mean fluorescence value of 9 (Fig. 1C).
Recombinant S. gordonii GP1422 is therefore a vaccine vector for a SIV antigen, also harboring OVA323-339 as a model peptide to assess in vivo the primary activation of antigen-specific CD4+ T cells using the adoptive transfer model system.
KJ1-26+ CD4+ T-cell distribution in mice immunized with recombinant S. gordonii GP1422. In order to assess the distribution of DO11.10 T cells following adoptive transfer, we tested the presence of transgenic cells in lymphoid organs of recipient BALB/c mice intranasally immunized with recombinant S. gordonii GP1422 and with the control strain GP1295. DO11.10 T cells, expressing the transgenic TCR that recognizes chicken OVA323-339 in the context of I-Ad (44), can be easily tracked in vivo by using monoclonal antibodies specific for CD4 and the clonotypic TCR (KJ1-26) (8).
In mice immunized with the control strain GP1295, the number of KJ1-26+ CD4+ T cells was very low in lymph nodes and spleen and almost undetectable in the NALT (Fig. 2), as observed in untreated animals (data not shown). The percentage of KJ1-26+ CD4+ T cells increased in the three lymphoid organs following immunization with recombinant bacteria carrying the OVA323-339 peptide. A time course analysis of the presence of KJ1-26+ CD4+ T cells in the NALT showed the highest percentage of transgenic cells 3 and 5 days after immunization (Fig. 2). The amount of KJ1-26+ CD4+ T cells in lymph nodes and spleen was smaller at day 3 and peaked at day 5. Among the three secondary lymphoid organs analyzed, the lowest number of transgenic KJ1-26+ CD4+ T cells was observed in the spleen (Fig. 2).
These data demonstrate that the clonal expansion of KJ1-26+ CD4+ T cells is antigen specific and not due to other vector antigens.
Antigen-specific T-cell activation in lymphoid organs. To study proliferation of DO11.10 T cells in vivo, transgenic cells were labeled with CFSE before injection into syngeneic recipient BALB/c mice. CFSE is a vital dye that can label cells very stably by covalently coupling to intracellular molecules, and it can be used to monitor lymphocyte proliferation due to the progressive halving of the dye fluorescence following cell division (19).
Mice inoculated with the control strain GP1295 maintained a high level of CFSE fluorescence in the three organs, indicating that no cell division had occurred (Fig. 3). As mentioned above, in the NALT of control mice the number of KJ1-26+ CD4+ T cells was undetectable. OVA-specific T cells proliferated in the NALT, lymph nodes, and spleens of mice immunized with the GP1422 strain carrying the OVA peptide, as shown by the progressive dilution of CFSE in the three organs (Fig. 3).
The time course analysis of the KJ1-26+ CD4+ T-cell activation performed 3, 5, and 8 days following immunization showed a high level of proliferation in the NALT from the third day, with 80% of proliferating cells; this reached almost 90% at day 5 and was maintained at the same values up to day 8 (Fig. 4A). In the draining lymph nodes and mainly in the spleen, the percentages of proliferating cells were lower at day 3 (57% and 14%, respectively) (Fig. 4B and C); they reached the peak of activation of approximately 90% in both organs at day 5 and declined at day 8. At day 5 approximately 90% of KJ1-26+ CD4+ T cells had divided between one and eight times in mice immunized with live GP1422 strain (Fig. 3). Inactivated recombinant S. gordonii also was able to stimulate a clear OVA-specific clonal expansion of KJ1-26+ CD4+ T cells (about 80% of transgenic dividing cells), although a higher proportion of still-undivided cells was present both in the lymph nodes and spleen (Fig. 3). When the PI was calculated, similar values were obtained for the NALTs of mice immunized with live and inactivated GP1422 (5 and 4.2, respectively). The PI was instead 1.85-fold higher in the lymph nodes and spleens of mice immunized with live bacteria (4.5 and 5.5, respectively) than in those treated with inactivated bacteria (2.4 and 3, respectively). Mice treated with the control strain GP1295 did not respond at any time points in any lymphoid organs tested (Fig. 4).
The delayed appearance of KJ1-26+ CD4+ T cells with diluted CFSE in lymph nodes and spleen following immunization suggests either a migration of activated antigen-specific CD4+ T cells from NALT to other secondary lymphoid organs or a delayed appearance of antigen-presenting cells loaded with S. gordonii migrating from the nose to the draining cervical lymph nodes.
DISCUSSION
The model of adoptive transfer of transgenic T lymphocytes (33) was used to demonstrate that intranasal immunization with the recombinant commensal bacterium S. gordonii is effective at inducing primary activation of antigen-specific CD4+ T cells in vivo, which is essential for the development of long-term immunity to vaccine antigens.
S. gordonii has been previously used as a vaccine vector for expression of a variety of antigens (25, 30), and it was shown to be immunogenic by systemic and mucosal routes both in mice and monkeys (6, 22-24, 26, 29, 31, 41). Furthermore, S. gordonii administered by the nasal/oral route has been recently shown to be safe in a phase I trial in 150 human volunteers, and it can be rapidly eradicated either spontaneously or with antibiotic treatment (14), suggesting that the use of S. gordonii is feasible in humans.
In the present study, a strong proliferative response of OVA-specific T cells was observed in the NALT, draining lymph nodes, and spleens of mice immunized with recombinant S. gordonii expressing the model fusion protein containing OVA. The cell activation observed towards the OVA T-helper epitope could be representative of CD4+ T-cell responses also induced towards T-helper epitopes of the model fusion protein. The highest levels of proliferation were observed 5 days following intranasal immunization, when about 90% of KJ1-26+ CD4+ T cells had divided between one and eight times. This is in accordance with what was observed with the human pathogen Streptococcus pyogenes administered by the nasal route when a comparable dose of inoculum was used (34). The antigen-specific CD4+ T-cell response was observed first in the NALT, already at 3 days following immunization, as previously shown with group A Streptococcus (34). This behavior is also similar to that observed in the gut following oral infection with Salmonella, which induces a T-cell response mainly in PP (21).
It is important to highlight that inactivated recombinant S. gordonii also was effective at inducing T-cell activation in the NALT and in the other secondary lymphoid organs analyzed; this is in agreement with recent data of ours indicating that inactivated recombinant S. gordonii expressing a meningococcal vaccine antigen is capable of inducing antigen-specific humoral responses following repeated intranasal immunizations (A. Ciabattini et al., unpublished data). These results open the potential of using inactivated recombinant commensal bacteria for intranasal immunization, thereby further circumventing the safety issues related to the use of live vaccine vectors.
Interestingly, in mice immunized with the wild-type strain, the number of OVA-specific CD4+ T cells (KJ1-26+) following adoptive transfer was low in lymph nodes and spleen and undetectable in the NALT, while it was increased in the three lymphoid organs following immunization with recombinant bacteria carrying the OVA323-339 peptide. This observation indicates that the KJ1-26+ CD4+ T-cell expansion is OVA specific and not due to vector antigens.
We have previously shown in vitro that human and mouse DCs process and present the vaccine antigens delivered by S. gordonii to CD4+ T cells much more efficiently than the soluble form (4, 39), and they also are more potent than B cells at presenting an MHC class II-restricted antigen expressed on the bacterial surface (5). As DCs have repeatedly been shown to be crucial for activation of naive T cells, we can therefore hypothesize that DCs present in the nasal mucosa (3, 9, 36) might be stimulated in vivo by recombinant S. gordonii, thereby inducing the efficient T-cell activation observed.
Repeated intranasal immunizations with recombinant S. gordonii were already shown to be effective at inducing local and systemic antibody responses capable of conferring protection (22). By using the adoptive transfer model system, we have demonstrated for the first time the efficient intranasal priming of naive antigen-specific CD4+ T cells against antigens expressed by live and inactivated recombinant S. gordonii in NALT, draining lymph nodes, and spleen. Taken together, these data further support the use of S. gordonii as an effective mucosal vaccine vector capable of inducing humoral and cellular immunity, both locally and systemically.
ACKNOWLEDGMENTS
We thank Riccardo Parigi for his skillful help with the animals.
This study was carried out with financial support from the Commission of the European Communities, Sixth Framework Programme, contract LSHP-CT-2003-503240, Mucosal Vaccines for Poverty-Related Diseases (MUVAPRED), and Fifth Framework Programme, contract QLK2-CT-2002-00882, Mucosal Vaccines Against Human and Simian Immunodeficiency Viruses Based on Dendritic Cells (MUVADEN) (to G.P.); from "Azione Concertata Italiana per lo Sviluppo di un Vaccino HIV/AIDS" (ICAV) of the Istituto Superiore di Sanita contract no. 45F.29 and MIUR (FIRB RBNE01RB9B_009) (to D.M.); and from the University of Minnesota School of Dentistry New Faculty Recruitment Fund (to M.C.).
REFERENCES
1. Asanuma, H., A. H. Thompson, T. Iwasaki, Y. Sato, Y. Inaba, C. Aizawa, T. Kurata, and S. Tamura. 1997. Isolation and characterization of mouse nasal-associated lymphoid tissue. J. Immunol. Methods 202:123-131.
2. Bergquist, C., E. L. Johansson, T. Lagergard, J. Holmgreen, and A. Rudin. 1997. Intranasal vaccination of humans with recombinant cholera toxin B subunit induces systemic and local antibody responses in the upper respiratory tract and the vagina. Infect. Immun. 65:2676-2684.
3. Bienenstock, J., and M. R. McDermott. 2005. Bronchus- and nasal-associated lymphoid tissue. Immunol. Rev. 206:22-31.
4. Corinti, S., D. Medaglini, A. Cavani, M. Rescigno, G. Pozzi, P. Ricciardi-Castagnoli, and G. Girolomoni. 1999. Human dendritic cells very efficiently present a heterologous antigen expressed on the surface of recombinant Gram-positive bacteria to CD4+ T lymphocytes. J. Immunol. 163:3029-3036.
5. Corinti, S., D. Medaglini, C. Prezzi, G. Pozzi, and G. Girolomoni. 2000. Human dendritic cells are superior to B cells at presenting a major histocompatibility complex class-II restricted heterologous antigen expressed on recombinant Streptococcus gordonii. Infect. Immun. 68:1879-1883.
6. Di Fabio, S., D. Medaglini, C. M. Rush, G. Corrias, G. L. Panzini, M. Pace, P. Verani, G. Pozzi, and F. Titti. 1998. Vaginal immunization of Cynomolgus monkeys with Streptococcus gordonii expressing HIV-1 and HPV 16 antigens. Vaccine 16:485-492.
7. Di Tommaso, A., G. Saletti, M. G. Pizza, R. Rappuoli, and G. Dougan. 1996. Induction of antigen-specific antibodies in vaginal secretions by using a nontoxic mutant of heat-labile enterotoxin as a mucosal adjuvant. Infect. Immun. 64:974-979.
8. Haskins, K., R. Kubo, J. White, M. Pigeon, J. Kappler, and P. Marrack. 1983. The major histocompatibility complex-restricted antigen receptor on T cells. I. Isolation with a monoclonal antibody. J. Exp. Med. 157:1149-1169.
9. Heritage, P. L., B. J. Underdown, A. L. Arsenault, D. P. Snider, and M. R. McDermott. 1997. Comparison of murine nasal-associated lymphoid tissue and Peyer's patches. Am. J. Respir. Crit. Care Med. 156:1256-1262.
10. Hirabayashi, Y., H. Kurata, H. Funato, T. Nagamine, C. Aizawa, S. Tamura, K. Shimada, and T. Kurata. 1990. Comparison of intranasal inoculation of influenza HA vaccine combined with cholera toxin B subunit with oral or parenteral vaccination. Vaccine 8:243-248.
11. Hiroi, T., K. Iwatani, H. Iijima, S. Kodama, M. Yanagita, and H. Kiyono. 1998. Nasal immune system: distinctive Th0 and Th1/Th2 type environments in murine nasal-associated lymphoid tissues and nasal passage, respectively. Eur. J. Immunol. 28:3346-3353.
12. Imaoka, K., C. J. Miller, M. Kubota, M. B. McChesney, B. Lohman, M. Yamamoto, K. Fujihashi, K. Someya, M. Honda, J. R. McGhee, and H. Kiyono. 1998. Nasal immunization of nonhuman primates with simian immunodeficiency virus p55gag and cholera toxin adjuvant induces Th1/Th2 help for virus-specific immune responses in reproductive tissues. J. Immunol. 161:5952-5958.
13. Kiyono, H., and S. Fukuyama. 2004. NALT- versus Peyer's-patch-mediated mucosal immunity. Nat. Rev. Immunol. 4:699-710.
14. Kotloff, K. L., S. S. Wassermann, K. F. Jones, S. Livio, D. E. Hruby, C. A. Franke, and V. A. Fischetti. 2005. Clinical and microbiological responses of volunteers to combined intranasal and oral inoculation with a Streptococcus gordonii carrier strain intended for future use as a group A Streptococcus vaccine. Infect. Immun. 73:2360-2366.
15. Kuper, C. F., P. J. Koornstra, D. M. H. Hameleers, J. Biewenga, B. J. Spit, A. M. Duijevestijn, P. J. C. van Breda Vriesman, and T. Sminia. 1992. The role of nasopharyngeal lymphoid tissue. Immunol. Today 13:219-223.
16. Kurono, Y., M. Yamamoto, K. Fujihashi, S. Kodama, M. Suzuki, G. Mogi, J. R. McGhee, and H. Kiyono. 1999. Nasal immunization induces Haemophilus influenzae-specific Th1 and Th2 responses with mucosal IgA and systemic IgG antibodies for protective immunity. J. Infect. Dis. 180:122-132.
17. Langermann, S., S. Palaszynski, A. Sadziene, C. K. Stover, and S. Koenig. 1994. Systemic and mucosal immunity induced by BCG vector expressing outer-surface protein A of Borrelia burgdorferi. Nature 372:552-555.
18. Lyons, A. B. 2000. Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution. J. Immunol. Methods 243:147-154.
19. Lyons, A. B., and C. R. Parish. 1994. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171:131-137.
20. Maggi, T., M. R. Spinosa, S. Ricci, D. Medaglini, G. Pozzi, and M. R. Oggioni. 2002. Genetic engineering of Streptococcus gordonii for the simultaneous display of two heterologous proteins at the bacterial surface. FEMS Microbiol. Lett. 210:135-141.
21. McSorley, S. J., S. Asch, M. Costalonga, R. L. Reinhardt, and M. K. Jenkins. 2002. Tracking Salmonella-specific CD4 T cells in vivo reveals a local mucosal response to a disseminated infection. Immunity 16:365-377.
22. Medaglini, D., A. Ciabattini, M. R. Spinosa, T. Maggi, H. Marcotte, M. R. Oggioni, and G. Pozzi. 2001. Immunization with recombinant Streptococcus gordonii expressing tetanus toxin fragment C confers protection from lethal challenge in mice. Vaccine 19:1931-1939.
23. Medaglini, D., M. R. Oggioni, and G. Pozzi. 1998. Vaginal immunization with recombinant Gram positive bacteria. Am. J. Reprod. Immunol. 39:199-208.
24. Medaglini, D., G. Pozzi, T. P. King, and V. A. Fischetti. 1995. Mucosal and systemic immune responses to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii after oral colonization. Proc. Natl. Acad. Sci. USA 92:6868-6872.
25. Medaglini, D., S. Ricci, T. Maggi, C. M. Rush, R. Manganelli, M. R. Oggioni, and G. Pozzi. 1997. Recombinant Gram-positive bacteria as vehicles of vaccine antigens. Biotechnol. Annu. Rev. 3:297-312.
26. Medaglini, D., C. M. Rush, P. Sestini, and G. Pozzi. 1997. Commensal bacteria as vectors for mucosal vaccines against sexually transmitted diseases: vaginal colonization with recombinant streptococci induces local and systemic antibodies in mice. Vaccine 15:1330-1337.
27. Muller, C. P., P. Beauverger, F. Schneider, F. Jung, and N. H. Brons. 1995. Cholera toxin B stimulates systemic neutralizing antibodies after intranasal co-immunization with measles virus. J. Gen. Virol. 76:1371-1380.
28. Murphy, K. M., A. M. Heimberger, and D. Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+ CD8+ TCRlo thymocytes in vivo. Science 250:1720-1723.
29. Oggioni, M. R., R. Manganelli, M. Contorni, M. Tommasino, and G. Pozzi. 1995. Immunization of mice by oral colonization with live recombinant commensal streptococci. Vaccine 13:775-779.
30. Oggioni, M. R., D. Medaglini, T. Maggi, and G. Pozzi. 1999. Engineering the Gram-positive cell surface for construction of bacterial vaccine vectors. Methods 19:163-173.
31. Oggioni, M. R., D. Medaglini, L. Romano, F. Peruzzi, T. Maggi, L. Lozzi, L. Bracci, M. Zazzi, F. Manca, P. E. Valensin, and G. Pozzi. 1999. Antigenicity and immunogenicity of the V3 domain of HIV-1 gp120 expressed on the surface of Streptococcus gordonii. AIDS Res. Hum. Retroviruses 15:451-459.
32. Oggioni, M. R., and G. Pozzi. 1996. A host-vector system for heterologous gene expression in Streptococcus gordonii. Gene 169:85-90.
33. Pape, K. A., E. R. Kearney, A. Khoruts, A. Mondino, R. Merica, Z. M. Chen, E. Ingulli, J. White, J. G. Johnson, and M. K. Jenkins. 1997. Use of adoptive transfer of T-cell-antigen-receptor-transgenic T cell for the study of T-cell activation in vivo. Immunol. Rev. 156:67-78.
34. Park, H. S., M. Costalonga, R. L. Reinhardt, P. E. Dombek, M. K. Jenkins, and P. P. Cleary. 2004. Primary induction of CD4 T cell responses in nasal associated lymphoid tissue during group A streptococcal infection. Eur. J. Immunol. 34:2843-2853.
35. Park, H. S., K. P. Francis, J. Yu, and P. P. Cleary. 2003. Membranous cells in nasal-associated lymphoid tissue: a portal of entry for the respiratory mucosal pathogen group A Streptococcus. J. Immunol. 171:2532-2537.
36. Porgador, A., H. F. Staats, Y. Itoh, and B. L. Kelsall. 1998. Intranasal immunization with cytotoxic T-lymphocyte epitope peptide and mucosal adjuvant cholera toxin: selective augmentation of peptide-presenting dendritic cells in nasal mucosa-associated lymphoid tissue. Infect. Immun. 66:5876-5881.
37. Pozzi, G., R. A. Musmanno, P. M. J. Lievens, M. R. Oggioni, P. Plevani, and R. Manganelli. 1990. Methods and parameters for genetic transformation of Streptococcus sanguis Challis. Res. Microbiol. 141:659-670.
38. Pozzi, G., M. R. Oggioni, R. Manganelli, D. Medaglini, V. A. Fischetti, D. Fenoglio, M. T. Valle, A. Kunkl, and F. Manca. 1994. Human T-helper cell recognition of an immunodominant epitope of HIV-1 gp120 expressed on the surface of Streptococcus gordonii. Vaccine 12:1071-1077.
39. Rescigno, M., S. Citterio, C. Thery, M. Rittig, D. Medaglini, G. Pozzi, S. Amigorena, and P. Ricciardi-Castagnoli. 1998. Bacterial-induced neo-biosynthesis, stabilization and surface expression of functional class I molecules in mouse dendritic cells. Proc. Natl. Acad. Sci. USA 95:5229-5234.
40. Rharbaoui, F., D. Bruder, M. Vidakovic, T. Ebensen, J. Buer, and C. A. Guzman. 2005. Characterization of a B220+ lymphoid cell subpopulation with immune modulatory functions in nasal-associated lymphoid tissues. J. Immunol. 174:1317-1324.
41. Ricci, S., D. Medaglini, C. M. Rush, A. Marcello, R. Manganelli, G. Palù, and G. Pozzi. 2000. Immunogenicity of the B monomer of the Escherichia coli heat-labile toxin expressed on the surface of Streptococcus gordonii. Infect. Immun. 68:760-766.
42. Shahin, R., M. Leef, J. Eldridge, M. Hudson, and R. Gilley. 1995. Adjuvanticity and protective immunity elicited by Bordetella pertussis antigens encapsulated in poly(DL-lactide-co-glycolide) microspheres. Infect. Immun. 63:1195-1200.
43. Shimoda, M., T. Nakamura, Y. Takahashi, H. Asanuma, S. Tamura, T. Kurata, T. Mizuochi, N. Azuma, C. Kanno, and T. Takemori. 2001. Isotype-specific selection of high affinity memory B cells in nasal-associated lymphoid tissue. J. Exp. Med. 194:1597-1607.
44. Shimonkevitz, R., S. Colon, J. W. Kappler, P. Marrack, and H. M. Grey. 1984. Antigen recognition by H-2-restricted T cells. II. A tryptic ovalbumin peptide that substitutes for processed antigen. J. Immunol. 133:2067-2074.
45. Spit, B. J., E. G. Hendriksen, J. P. Bruijntjes, and C. F. Kuper. 1989. Nasal lymphoid tissue in the rat. Cell Tissue Res. 255:193-198.
46. Unger, W. W., W. Jansen, D. A. Wolvers, A. G. van Halteren, G. Kraal, and J. N. Samsom. 2003. Nasal tolerance induces antigen-specific CD4+CD25– regulatory T cells that can transfer their regulatory capacity to naive CD4+ T cells. Int. Immunol. 15:731-739.
47. Waldo, F. B., A. W. van den Wall Bake, J. Mestecky, and S. Husby. 1994. Suppression of the immune response by nasal immunization. Clin. Immunol. Immunopathol. 72:30-34.
48. Wu, H. Y., H. H. Nguyen, and M. W. Russel. 1997. Nasal lymphoid tissue (NALT) as a mucosal immune inductive site. Scand. J. Immunol. 46:506-513.
49. Wu, H. Y., E. B. Nikolova, K. W. Beagley, and M. W. Russell. 1996. Induction of antibody-secreting cells and T-helper and memory cells in murine nasal lymphoid tissue. Immunology 88:493-500.
50. Wu, H. Y., and M. W. Russel. 1997. Nasal lymphoid tissue, intranasal immunization, and compartmentalization of the common mucosal immune system. Immunol. Res. 16:187-201.
51. Wu, H. Y., and M. W. Russel. 1993. Induction of mucosal immunity by intranasal application of a streptococcal surface protein antigen with the cholera toxin B subunit. Infect. Immun. 61:314-322.
52. Yanagita, M., T. Hiroi, N. Kitagaki, S. Hamada, H. O. Ito, H. Shimauchi, S. Murakami, H. Okada, and H. Kiyono. 1999. 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. 162:3559-3565.
53. Zuercher, A. W. 2003. Upper respiratory tract immunity. Viral Immunol. 16:279-289.
54. Zuercher, A. W., S. E. Coffin, M. C. Thurnheer, P. Fundova, and J. J. Cebra. 2002. Nasal-associated lymphoid tissue is a mucosal inductive site for virus-specific humoral and cellular immune responses. J. Immunol. 168:1796-1803.(Donata Medaglini, Annalis)