The Nasal Dendritic Cell-Targeting Flt3 Ligand as a Safe Adjuvant Elicits Effective Protection against Fatal Pneumococcal Pneumonia

Abstract

We have previously shown that a pneumococcal surface protein A (PspA)-based vaccine containing DNA plasmid encoding the Flt3 ligand (FL) gene (pFL) as a nasal adjuvant prevented nasal carriage of Streptococcus pneumoniae. In this study, we further investigated the safety and efficacy of this nasal vaccine for the induction of PspA-specific antibody (Ab) responses against lung infection with S. pneumoniae. C57BL/6 mice were nasally immunized with recombinant PspA/Rx1 (rPspA) plus pFL three times at weekly intervals. When dynamic translocation of pFL was initially examined, nasal pFL was taken up by nasal dendritic cells (DCs) and epithelial cells (nECs) but not in the central nervous systems, including olfactory nerve and epithelium. Of importance, nasal pFL induced FL protein synthesis with minimum levels of inflammatory cytokines in the nasal washes (NWs) and bronchoalveolar lavage fluid (BALF). NWs and BALF as well as plasma of mice given nasal rPspA plus pFL contained increased levels of rPspA-specific secretory IgA and IgG Ab responses that were correlated with elevated numbers of CD8⁺ and CD11b⁺ DCs and interleukin 2 (IL-2)- and IL-4-producing CD4⁺ T cells in the nasal mucosa-associated lymphoid tissues (NALT) and cervical lymph nodes (CLNs). The in vivo protection by rPspA-specific Abs was evident in markedly reduced numbers of CFU in the lungs, airway secretions, and blood when mice were nasally challenged with Streptococcus pneumoniae WU2. Our findings show that nasal pFL is a safe and effective mucosal adjuvant for the enhancement of bacterial antigen (Ag) (rPspA)-specific protective immunity through DC-induced Th2-type and IL-2 cytokine responses.

INTRODUCTION

Streptococcus pneumoniae is a leading human pathogen causing diseases ranging from otitis media to pneumonia, bacteremia, and meningitis. This bacterium, commonly termed the pneumococcus, can result in an estimated 1.6 million deaths per year worldwide, more than half of which are young children in developing countries (2). Although pneumococcal capsular polysaccharide and pneumococcal protein-capsular conjugate vaccines can provide protective immunity against pneumonia and invasive diseases in adults and infants, a strong need still exists for a new generation of effective vaccines for the prevention of all potential S. pneumoniae infections. In this regard, the multivalent polysaccharide vaccines do not provide protection against strains with nonvaccine serotypes (28, 41). Of importance, pneumococcal surface protein A (PspA) has been extensively investigated as a candidate vaccine antigen (Ag) to prevent pneumococcal infection (5, 37). For instance, PspA-specific antibody (Ab) enhances bacterial clearance and induces cross-protection against infection with strains of different serotypes (4, 31). Further, previous studies have demonstrated that PspA-specific Abs overcome the anticomplementary effect of PspA, allowing increased complement activation and C3 deposition on PspA-bearing bacteria (27, 30).

Nasal immunization has been shown to preferentially induce Ag-specific Ab responses in the respiratory tract (20) and other mucosal lymphoid tissues (10, 25, 26). To induce maximal levels of Ag-specific immune responses in both mucosal and systemic lymphoid tissue compartments, it is often necessary to use a mucosal adjuvant (16, 22, 39). Although native cholera toxin and related Escherichia coli enterotoxin are potent mucosal adjuvant for enhancement of Ag-specific immune responses, their application for human use is not warranted since they can cause diarrhea or Bell's palsy (6, 23, 29). Moreover, these toxins are known to migrate into and accumulate in the olfactory tissues when given nasally (40). In this regard, our previous studies demonstrated that nasal application of a DNA plasmid (pFL) containing the gene of the Flt3 ligand (FL), which is a kind of cytokine, preferentially expanded CD8⁺ dendritic cells (DCs) and subsequently induced Ag-specific mucosal immune responses mediated by interleukin 4 (IL-4)-producing CD4⁺ T cells when mice were nasally administrated ovalbumin with pFL as the mucosal adjuvant (19). Further, a combination of nasal pFL and CpG oligonucleotides as a double DNA adjuvant enhanced mucosal and systemic immune responses via induction of plasmacytoid DCs as well as CD8⁺ DCs in mucosal compartments (11, 17). Nasal administration of an adenovirus vector encoding FL cDNA also showed enhancement and maintenance of long-term immunity (17, 32).

In this study, we examined the safety and effectiveness of nasal pFL as a mucosal adjuvant for the induction of functional bacterial Ag (recombinant PspA [rPspA])-specific Ab responses for protection against S. pneumoniae infection in the lower respiratory tract. Our findings show that nasal rPspA plus pFL adjuvant successfully elicits protective immunity in both the upper and lower respiratory tracts by enhancing mucosal DC-mediated Th2-type and IL-2 cytokine responses without detectable cytokine-mediated inflammation.

MATERIALS AND METHODS

Mice.

Specific-pathogen-free female C57BL/6 mice (6 to 8 weeks old) were purchased from Charles River Japan (Kanagawa, Japan) and used in this study. Upon arrival, these mice were transferred to microisolators, maintained in horizontal laminar flow cabinets, and provided sterile food and water as part of a specific-pathogen-free facility at Osaka University (Suita, Japan), and all experiments were conducted in accordance with the guidelines provided by Osaka University. All of the mice used in these assays were free of bacterial and viral pathogens.

rPspA and adjuvants.

Endotoxin-free rPspA was purified by chromatography on a chelating Sepharose 4B column preloaded with Ni⁺ (GE Healthcare, Piscataway, NJ) from Escherichia coli BL21(DE3) carrying pUAB055, which comprised the first 302 of the 588 amino acids of PspA/Rx1, including all of the α-helical region and some of the proline-rich region (3). The plasmid pORF9-mFlt3L (pFL) consists of the pORF9-mcs vector (pORF) plus the full-length murine FL cDNA gene (InvivoGen, San Diego, CA). These plasmids were purified using the Gene Elute endotoxin-free plasmid kit (Sigma-Aldrich, St. Louis, MO) (19). The Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD) resulted in <0.1 endotoxin unit of lipopolysaccharide (LPS) per 1 μg of plasmids or rPspA.

Nasal immunization and sample collection.

Mice were immunized three times at weekly intervals nasally with 6 μl/nostril phosphate-buffered saline (PBS) containing 5 μg of rPspA and 50 μg of pFL as a mucosal adjuvant. As controls, mice were immunized nasally with 50 μg of pORF (empty plasmid) and 5 μg of rPspA under anesthesia. In some experiments, mice were administered pFL (50 μg), pORF (50 μg), rPspA (1 μg or 5 μg), native cholera toxin (nCT) (1 μg), or PBS alone under anesthesia. Plasma, nasal washes (NWs), and bronchoalveolar lavage fluid (BALF) were obtained as described previously (36).

Dynamic translocation of pFL.

On 12 h or 7 days after mice were nasally given pFL (50 μg) alone, mononuclear cells were isolated from nasal mucosa-associated lymphoreticular tissues (NALT) and nasal passages (NPs) as described previously (14, 19), and NALT and NP dendritic cells (DCs) were purified by the AutoMACS cell sorter (Miltenyi Biotec, Auburn, CA) using anti-CD11c monoclonal Ab (MAb) microbeads (19). Further, nasal epithelial cells (nECs) and olfactory nerves and epithelium (ON/E) were isolated from nasal passages and olfactory bulbs, respectively (14, 40). In brief, cells from the nasal mucosa and olfactory bulb were prepared by gentle teasing through stainless screens and were subjected to discontinuous gradient centrifugation using 40% and 55% Percoll. Cells on the surface of the 40% layer were used as nECs and ON/E. To further confirm the presence of nECs and ON/E, the size and granularity of cells were determined by using flow cytometry. DNA was then extracted from NALT, NP-DCs, nECs, and ON/E, and the ampicillin resistance gene (858 bp) contained in the pFL plasmid was detected by a primer-specific PCR method. The sense primer was 5′-CCA ATG CTT AAT CAG TGA GGC-3′, and the anti-sense primer was 5′-ATG AGT ATT CAA CAT TTC CGT GTC G-3′. The PCR products were separated by electrophoresis in 2% agarose gels and visualized by UV light illumination following ethidium bromide (0.5 mg/ml) staining (19).

Analysis of FL protein synthesis.

Twelve hours after nasal administration of pFL (50 μg), empty plasmid (50 μg), rPspA alone, or PBS, DCs from NALT and NPs and nECs and ON/E were purified aseptically as described above and were then cultured for 48 h (2 × 10⁶ cells/ml) in complete medium. The concentrations of FL protein secreted into the medium were determined by FL-specific enzyme-linked immunosorbent assay (ELISA) (Quantikine M mouse Flt3 ligand ELISA kit; R & D Systems, Minneapolis, MN). Mice were next nasally immunized weekly for three consecutive weeks with rPspA (5 μg) plus pFL (50 μg) or pORF (50 μg), rPspA alone (5 μg), or PBS, and 1 week after the last immunization, the FL protein in nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) was determined by FL-specific ELISA (R & D Systems).

Detection of inflammatory cytokines in mucosal secretion.

In order to determine inflammatory cytokines by nasal application of pFL, NWs and BALF were collected 5 days after the nasal administration of pFL (50 μg), pORF (50 μg), rPspA (1 μg or 5 μg), or native cholera toxin (1 μg). Next, the mucosal secretion samples were subjected to ELISA specific to IL-1β, IL-6 (R & D Systems), and tumor necrosis factor alpha (TNF-α) according to the manufacturer's instructions (eBioscience, San Diego, CA).

rPspA-specific Ab assays.

In order to examine mucosal and systemic immune responses to Ag, rPspA-specific IgA and IgG antibody (Ab) levels in plasma, NWs, and BALF were determined by ELISA on day 7 after the last immunization, as described previously (18, 19, 32). Briefly, 96-well Falcon microtest assay plates (BD Biosciences, Oxnard, CA) were coated with 1 μg/ml of rPspA in PBS. After incubating serial dilutions of samples, horseradish peroxidase-conjugated goat anti-mouse IgM, IgG, IgG1, IgG2a, IgG2b, IgG3, or IgA (Southern Biotechnology Associates Inc., Birmingham, AL) was added to wells. The color reaction was developed for 15 min at room temperature. Endpoint titers were expressed as the reciprocal log₂ of the last dilution that gave an optical density at 415 nm (OD₄₁₅) of 0.1 greater than the background level. Further, mononuclear cells obtained from spleen, NALT, cervical lymph nodes (CLNs), mediastinal lymph nodes (MeLNs), NPs, and lungs were subjected to an enzyme-linked immunospot (ELISPOT) assay in order to determine the numbers of Ag-specific Ab-forming cells (AFCs) (18, 19). In brief, mononuclear cells in the spleen, NALT, CLNs, and MeLNs were isolated aseptically by a mechanical dissociation method using gentle teasing through stainless steel screens as described previously (14). For isolation of mononuclear cells from NPs, a modified dissociation method was used based upon a previously described protocol (18). Mononuclear cells from lungs were isolated by a combination of an enzymatic dissociation procedure with collagenase type IV (0.5 mg/ml; Sigma-Aldrich) followed by discontinuous Percoll (Amersham Biosciences, Arlington Heights, IL) gradient centrifugation.

Flow cytometric analysis.

To characterize the phenotype of DCs, aliquots of mononuclear cells (0.2 × 10⁶ to 1.0 × 10⁶ cells) were isolated from various lymphoid compartments 1 week after the last immunization with rPspA plus pFL or pORF. The cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD11b, CD8, or B220 MAbs (BD Biosciences). In some experiments, mononuclear cells were incubated with phycoerythrin (PE)-labeled anti-mouse I-A^b, CD11c, CD40, CD80, or CD86 MAbs (BD Biosciences) and biotinylated anti-mouse CD11c MAbs (BD Biosciences), followed by CyChrome-streptavidin. These samples were then subjected to flow cytometry analysis (FACSCalibur; BD Biosciences) for cell subset analysis (19).

rPspA-specific CD4⁺ T cell responses and cytokine-specific ELISA.

CD4⁺ T cells from lungs, CLNs, and spleen were purified using an automatic cell sorter (AutoMACS) system (Miltenyi Biotec) as described previously (18, 19). The purified CD4⁺ T cell fraction (>97% CD4⁺ and >99% viable) was resuspended in RPMI 1640 (Sigma-Aldrich) supplemented with HEPES buffer (10 mM), l-glutamine (2 mM), nonessential amino acid solution (10 μl/ml), sodium pyruvate (10 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), gentamicin (80 μg/ml), and 10% fetal calf serum (FCS) (complete medium; 4 × 10⁶ cells/ml) and cultured in the presence of T cell-depleted, complement- and mitomycin-treated splenic Ag-presenting cells taken from nonimmunized, normal mice with or without 2 μg/ml rPspA. To assess rPspA-specific T cell proliferative responses, an aliquot of 0.5 μCi of tritiated [³H]TdR (PerkinElmer Japan Co., Ltd., Japan) was added during the final 18 h of incubation, and the amount of [³H]TdR incorporation was determined by scintillation counting (19). The culture supernatants were collected on day five and analyzed using gamma interferon (IFN-γ)-, IL-2-, IL-4-, IL-5-, IL-6-, and IL-10-specific ELISA kits (eBioscience). The detection limit for each cytokine was as follows: 15 pg/ml for IFN-γ, 2 pg/ml for IL-2, 4 pg/ml for IL-4, IL-5, and IL-6, and 30 pg/ml for IL-10.

Pneumococcal infection.

Mice were nasally challenged with a serotype 3 S. pneumoniae strain (WU2) with a mucoid phenotype at a dose of 1.8 × 10⁷ CFU (20 μl). Forty-eight hours after the bacterial challenge, the lungs were removed aseptically and homogenized in 9 ml of sterile saline per gram of lung tissues. NWs and blood were collected as described above. Bacterial colonies were counted by plating lungs, NWs, and blood (50 μl, respectively) on horse blood agar (BD Biosciences), followed by incubation at 37°C overnight. The detection limit of bacterial culture was 10² CFU/g. The 50% lethal dose was calculated to be 2.5 × 10⁶ CFU.

Statistical analysis.

Each result is expressed as the mean ± 1 standard error of the mean (SEM). All mouse groups were compared with control mice using an unpaired Mann-Whitney U test by using the Statview software program (Abacus Concepts, Cary, NC), designed for Macintosh computers, with Bonferroni's correction. P values of <0.05 or <0.01 were considered significant.

RESULTS

Tracking plasmid expression and FL protein synthesis.

In order to examine safety of pFL for nasal application, we initially traced plasmid-specific ampicillin resistance gene expression by nasal DCs, nECs, and the ON/E. DCs from NALT and NPs, as well as nECs, possessed the ampicillin resistance gene 12 h after nasal administration of pFL (Fig. 1A, left). Of interest, on 7 days after nasal pFL application, the ampicillin resistance gene was detected only in nECs (Fig. 1A, right). Further, NALT-DCs, NP-DCs, and nECs of mice given nasal pFL produced significantly elevated levels of the FL protein compared with those of mice given nasal pORF (empty plasmid), rPspA alone, or PBS (Fig. 1B). In addition, nasal application of the combination of rPspA and pFL resulted in FL protein production comparable to that with nasal application of pFL alone (data not shown). However, FL protein synthesis in mice given nasal rPspA plus pORF was at essentially the same level as that seen in mice given pORF or rPspA alone (data not shown). Thus, NWs and BALF from mice given nasal pFL plus rPspA contained significantly higher levels of FL than those from mice given nasal pORF plus rPspA, rPspA alone, or PBS only (Fig. 1C). On the other hand, of importance, no plasmid-specific genes were essentially detected in the ON/E of mice given nasal pFL (Fig. 1A). Thus, the culture supernatants of ON/E did not contain detectable levels of the FL protein (Fig. 1B). These results show that pFL is largely present in nasal DCs and nECs but not in the ON/E and suggest that pFL on nECs may maintain production of the FL protein.

Fig. 1.

(A to C) Translocation of FL plasmid after nasal administration of pFL (A), FL protein production by nasal DCs and epithelial cells (B), and expression of the FL protein in mucosal secretions (C). (A) Twelve hours (left) or 7 days (right) after nasal application of pFL (50 μg), the DNA samples were extracted from 1.0 × 10⁵ (each) cells of the olfactory nerve and epithelium (ON/E; lane 2), NALT-DCs (lane 3), NPs-DCs (lane 4), and nasal epithelial cells (nECs; lane 5). In order to show the presence of plasmid in these cell populations, the ampicillin resistance gene (858 bp) contained in pFL was detected by PCR using specific primers. pFL (0.1 μg) was employed as a positive control (lane 1). (B) Mice were nasally administered pFL (50 μg; black column), pORF (50 μg; white column), rPspA (5 μg; shaded column), or PBS (hatched column). Twelve hours later, NALT-DCs, NPs-DCs, nECs, and ON/E were isolated and cultured (2 × 10⁶ cells/ml, respectively) for 48 h in complete medium. The concentration of FL protein secreted in medium was measured by FL-specific ELISA. The values shown are the means ± SEM for 30 mice for each group and a total of three experiments. *, P < 0.05 compared with results for the mouse group given pORF, rPspA, or PBS. (C) Mice were nasally immunized weekly for three consecutive weeks with rPspA (5 μg) plus pFL (50 μg; black column) or pORF (50 μg; white column), rPspA alone (5 μg; shaded column), or PBS (hatched column). One week after the last immunization, NWs and BALF (100 μl, respectively) were collected and subjected to FL-specific ELISA. The values shown are the means ± SEM of data for 30 mice for each group and a total of three experiments. *, P < 0.05 compared with results for mouse group given pORF, rPspA, or PBS.

Nasal pFL induces lower levels of inflammatory cytokines than nCT.

Although pFL was not taken up by the central nervous system, it is important to show that FL produced in the nasal cavity does not induce inflammatory responses. In this regard, the levels of IL-1β, IL-6, and TNF-α production in NWs and BALF were examined 5 days after nasal administration with rPspA, pORF, native cholera toxin (nCT), or pFL. The levels of inflammatory cytokine synthesis in NWs and BALF of mice given nasal pFL were essentially the same as or lower than that of mice given nasal rPspA or pORF alone (Fig. 2). Similarly, nasal application of rPspA plus pFL resulted in low levels of inflammatory cytokine production which were similar to those seen in pFL alone (data not shown). Conversely, nasal nCT induced markedly high levels of these inflammatory cytokines (Fig. 2). These results show that nasal pFL application does not elicit unnecessary inflammatory responses in the nasal mucosa.

Fig. 2.

Inflammatory cytokine production in NWs (white column) and BALF (black column). Mice were nasally administered native cholera toxin (nCT) (1 μg), rPspA (1 or 5 μg), pORF (50 μg), or pFL (50 μg). Five days later, NWs and BALF were collected and subjected to IL-1β-, IL-6-, and TNF-α-specific ELISA. The values shown are the means ± SEM of data for 30 mice for each group and total of three experiments. *, P < 0.05; **, P < 0.01 (compared with results for mouse group given nCT).

Induction of rPspA-specific Ab responses in mucosal and systemic tissues of mice given rPspA plus pFL.

We next examined whether nasal administration of pFL as a mucosal adjuvant would enhance rPspA-specific Ab responses. Giving mice nasal rPspA plus pFL resulted in significantly increased levels of rPspA-specific IgA Ab responses in NWs and BALF compared with results for mice given nasal rPspA plus pORF or rPspA Ag alone (Fig. 3A). The levels of rPspA-specific IgA Ab responses for mice given rPspA plus pFL were comparable to those seen for mice given nasal nCT vaccination (Fig. 3A). To further support these findings, elevated numbers of PspA-specific IgA AFCs were detected in NPs, CLNs, NALT, lungs, and MeLNs of mice given nasal rPspA and pFL (Fig. 3B). In addition, significantly higher numbers of rPspA-specific IgG and/or IgM AFCs were seen for mice given pFL as a nasal adjuvant than for mice given nasal pORF or rPspA alone (Fig. 3B). These results clearly show that pFL as a nasal adjuvant effectively elicits rPsp-specific Ab responses in mucosa-associated lymphoid tissues in the respiratory tract. Since nasal immunization is known to induce systemic immunity in addition to the mucosa, rPspA-specific Ab responses in plasma and spleen were examined. Nasal pFL as a mucosal adjuvant successfully enhanced rPspA-specific IgG and IgA Ab responses in plasma which are comparable to those responses seen in mice given nasal rPspA plus nCT (Fig. 4A). Thus, significantly increased numbers of rPspA-specific IgM, IgG, and IgA AFCs were seen in spleen of mice given pFL as a nasal adjuvant (Fig. 4B). When the levels of rPspA-specific IgG subclass Ab responses were examined, increased levels of anti-rPspA IgG1, IgG2a, and IgG2b Abs were noted for mice given nasal rPspA plus pFL compared with those Ab responses for mice given rPspA plus pORF or rPspA alone (Fig. 4A). Essentially no IgG3 Ab response against rPspA was detected. Taken together, pFL as a nasal adjuvant effectively induces rPspA-specific Ab responses in both mucosal and systemic immune compartments.

Fig. 3.

Mucosal immune responses to rPspA in external secretions and mucosal lymphoid tissues. C57BL/6 mice were nasally immunized three times at weekly intervals with rPspA (5 μg) plus pFL (50 μg; black column) or pORF (50 μg; white column), rPspA alone (shaded column), or rPspA (5 μg) plus nCT (1 μg; hatched column). (A) Seven days after the last immunization, the levels of rPspA-specific IgA Abs in NWs and BALF were determined by rPspA-specific ELISA. (B) Seven days after the last immunization, mononuclear cells isolated from NPs, CLNs, NALT, lungs, and MeLNs were subjected to ELISPOT assay to determine the numbers of Ag-specific IgM, IgG, and IgA Ab-forming cells (AFCs). The values shown are the means ± SEM (n = 20). *, P < 0.05; **, P < 0.01 (compared with mouse group given rPspA alone). †, P < 0.05 (compared with mouse group given rPspA plus pORF).

Fig. 4.

Comparison of rPspA-specific Ab responses in plasma and spleen cells of mice given nasal rPspA plus pFL (black column) or pORF (white column), rPspA alone (shaded column), or rPspA plus nCT (hatched column). Each mouse group was nasally immunized weekly for three consecutive weeks. (A) Seven days after the last immunization, rPspA-specific IgM, IgG, IgA, and IgG subclass Ab responses in plasma were determined by Ag-specific ELISA. An rPspA-specific IgG3 Ab response was not detected. (B) Seven days after the last immunization, mononuclear cells were isolated from spleens and were then subjected to ELISPOT assay to determine numbers of rPspA-specific IgM, IgG, and IgA AFCs. The values shown are the means ± SEM (n = 20). *, P < 0.05; **, P < 0.01 (compared with results for mouse group given rPspA alone). †, P < 0.05 (compared with results for mouse group given rPspA plus pORF).

Nasal rPspA plus pFL leads CD11b⁺ and CD8⁺ DCs.

Since our previous studies reported that nasal pFL plus ovalbumin as an Ag elicited expansion of CD8-expressing lymphoid-type CD11c⁺ DCs (19), we next characterized CD11c⁺ DCs in the various mucosal tissues of mice given rPspA plus pFL or pORF. Nasal immunization of rPspA plus pFL significantly increased the frequency of CD11c⁺ cells in both mucosal and systemic tissues compared with results for mice given rPspA plus pORF (Table 1). Interestingly, the numbers of CD8⁺ DCs were increased in all tissues of mice given pFL as a nasal adjuvant compared with those numbers for mice given nasal pORF. In addition, increased frequencies of CD11b⁺ DCs were noted in NALT, NPs, CLNs, and spleen. In contrast, increased frequencies of B220⁺ DCs were seen only in CLNs (Table 1). Further, higher expression of major histocompatibility complex class II (MHC II), CD40, CD80, and CD86 was seen on CD11c⁺ DCs (Table 1). CD8⁺ and CD11b⁺ DCs from NALT, lungs, and NPs were also examined by fluorescence-activated cell sorting (FACS) for expression of costimulatory molecules. Our results showed increased frequencies of costimulatory molecule expression by CD8⁺ and CD11b⁺ DCs in the tissues of mice given nasal pFL compared with those in control groups (Table 2). Taken together, these results indicate that nasal administration of rPspA plus pFL preferentially expands the numbers of CD8⁺ and CD11b⁺ DC populations which express elevated levels of costimulatory molecules.

Table 1.

Frequencies of CD11c⁺ DCs and CD8, CD11b, B220, and costimulatory molecule expression by CD11c⁺ DCs of mucosal effector and inductive tissues of mice given nasal rPspA with pFL or pORF^a,e

Tissue source	Adjuvant given with nasal rPspA	% CD11c+ total lymphocytesb	% CD11c+ DCs expressing:
			CD8c	CD11bc	B220c	CD40d	CD80d	CD86d	MHC IId
NALT	pFL	*6.6 ± 1.7	*23.5 ± 3.7	*23.1 ± 3.1	56.5 ± 6.2	**3.1 ± 0.7	10.9 ± 4.5	*19.3 ± 4.8	**68.9 ± 8.2
	pORF	1.7 ± 1.2	12.1 ± 1.6	12.3 ± 3.9	50.4 ± 0.6	0.4 ± 0.5	5.9 ± 1.6	8.9 ± 2.6	45.9 ± 1.9
Lungs	pFL	*5.7 ± 1.3	*12.4 ± 2.4	68.6 ± 4.1	24.8 ± 3.5	*4.6 ± 1.1	*15.2 ± 5.6	*20.8 ± 3.6	*51.5 ± 9.6
	pORF	3.3 ± 1.6	7.7 ± 0.9	63.9 ± 3.9	20.6 ± 4.7	1.6 ± 0.1	6.9 ± 2.4	9.5 ± 7.9	39.1 ± 0.7
NPs	pFL	*10.1 ± 2.4	*22.1 ± 2.3	*60.1 ± 6.9	19.3 ± 3.7	*11.1 ± 3.3	*23.8 ± 3.5	25.9 ± 6.2	38.5 ± 4.2
	pORF	5.3 ± 0.9	14.8 ± 3.8	31.1 ± 6.0	20.6 ± 4.7	3.6 ± 0.7	17.8 ± 0.1	23.1 ± 7.6	33.3 ± 1.9
CLNs	pFL	*2.3 ± 0.4	*32.8 ± 3.6	*31.8 ± 3.6	*45.4 ± 4.1	**3.1 ± 0.2	**24.7 ± 1.0	*43.3 ± 2.9	87.4 ± 5.9
	pORF	0.7 ± 0.2	21.1 ± 3.6	24.8 ± 3.2	33.3 ± 4.5	0.7 ± 0.9	10.6 ± 1.3	28.5 ± 8.5	85.3 ± 0.6
Spleen	pFL	*2.4 ± 0.7	*21.7 ± 2.2	*33.4 ± 5.1	44.6 ± 3.5	*4.3 ± 2.0	20.3 ± 6.4	**26.4 ± 5.3	**87.5 ± 1.5
	pORF	1.4 ± 0.5	18.2 ± 0.9	25.2 ± 3.4	39.9 ± 3.5	1.1 ± 0.1	12.9 ± 0.6	11.4 ± 2.8	75.0 ± 4.2

^aMononuclear cells from NALT, lungs, NPs, CLNs, and spleens of mice immunized with rPspA plus pFL or pORF were stained with a combination of anti-CD11c and the respective MAb and subjected to FACSCalibur flow cytometry analysis.

^bMononuclear cells were stained with PE-conjugated anti-CD11c MAb and subjected to flow cytometry analysis.

^cMononuclear cells were stained with FITC-conjugated anti-CD8α, -CD11b, and -B220 MAb and PE-labeled anti-CD11c.

^dMononuclear cells were stained with PE-labeled anti-CD40, -CD80, -CD86 or I-A and biotinylated anti-mouse CD11c MAb followed by CyChrome-streptavidin.

^e*, P < 0.05; **, P < 0.01 (compared with those of mice immunized with rPspA plus pORF).

Table 2.

Frequencies of costimulatory molecule expression on CD8 or CD11b DCs in mucosal effector and inductive tissues of mice given nasal rPspA with pFL or pORF^a

Tissue source	Adjuvant given with nasal rPspA	% CD11c+ DCs
		CD8+ DCs				CD11b+ DCs
		CD40	CD80	CD86	MHC II	CD40	CD80	CD86	MHC II
NALT	pFL	**1.4 ± 0.1	*4.4 ± 0.6	*9.5 ± 1.4	**34.5 ± 2.2	**1.3 ± 0.1	*4.1 ± 0.3	*8.5 ± 1.7	*27.3 ± 4.2
	pORF	0.2 ± 0.1	2.0 ± 0.4	4.0 ± 0.7	12.4 ± 1.3	0.2 ± 0.1	1.7 ± 0.2	3.7 ± 1.0	14.6 ± 1.5
Lungs	pFL	*1.7 ± 0.2	**7.7 ± 0.3	**7.5 ± 0.4	**18.7 ± 3.1	*1.4 ± 0.2	*6.6 ± 0.9	*8.0 ± 0.8	*30.5 ± 3.8
	pORF	0.5 ± 0.1	1.3 ± 0.1	2.3 ± 0.2	6.2 ± 1.0	0.9 ± 0.3	3.5 ± 0.9	3.6 ± 1.0	14.5 ± 5.7
NPs	pFL	*5.8 ± 0.4	*8.2 ± 1.7	*9.8 ± 1.5	*14.8 ± 2.4	*4.5 ± 1.1	*13.1 ± 2.0	11.5 ± 3.3	*20.6 ± 3.5
	pORF	1.4 ± 0.3	4.9 ± 1.4	4.9 ± 1.0	7.3 ± 1.9	2.5 ± 1.0	7.0 ± 1.7	8.7 ± 2.0	12.0 ± 2.2

^aCD11c-positive DCs from NALT, lungs, and NPs of mice immunized with rPspA plus pFL or pORF were purified from mononuclear cells by using the automatic cell sorter system AutoMacs and were stained with a combination of FITC-conjugated anti-mouse CD8α MAb or anti-mouse CD11b MAb and PE-labeled anti-CD40, -CD80, -CD86, or I-A and subjected to FACSCalibur flow cytometry analysis. *, P < 0.05; **, P < 0.01 (compared with those of mice immunized with rPspA plus pORF).

Th1- and Th2-type cytokine responses by PspA-specific CD4⁺ T cells.

We next assessed rPspA-specific CD4⁺ T cell responses induced by pFL as a mucosal adjuvant. rPspA-stimulated CD4⁺ T cells from lungs, CLNs, and spleen of mice given nasal rPspA plus pFL showed significantly higher proliferative responses than did those from mice nasally immunized with rPspA plus pORF (Table 3). In this regard, when Th1- and Th2-type cytokine profiles were examined, PspA-stimulated CD4⁺ T cells from mice given pFL as a nasal adjuvant exhibited higher levels of IL-2, IL-4, IL-5, and IL-6 production than those in control mice. On the other hand, levels of IFN-γ production by PspA-stimulated CD4⁺ T cells were essentially the same between mice given pFL or pORF (Table 3). Intracellular IL-17 analysis revealed that no significant increase in the frequency of IL-17-producing CD4⁺ T cells was seen in CLNs and spleen of mice given nasal pFL compared with results for mice given empty plasmid as a nasal adjuvant (data not shown). These results show that pFL as a nasal adjuvant preferentially induces Th2-type dominant cytokine responses in the lower respiratory mucosa when rPspA is used as an Ag for nasal vaccination.

Table 3.

CD4⁺ Th1- and Th2-type cytokine responses after in vitro restimulation with rPspA^a

Tissue	Nasal adjuvant	Stimulation indexb	Production of Th1- or Th2-type cytokinec
			IFN-γ (ng/ml)	IL-2 (pg/ml)	IL-4 (pg/ml)	IL-5 (pg/ml)	IL-6 (ng/ml)	IL-10 (ng/ml)
Lungs	pFL	*5.5 ± 1.8	2.03 ± 0.40	*249.8 ± 48.8	*56.7 ± 9.4	*255.7 ± 76.9	*1.80 ± 0.49	1.19 ± 0.32
	pORF	2.5 ± 0.6	1.48 ± 0.24	130.1 ± 35.7	21.2 ± 4.4	86.6 ± 22.0	0.78 ± 0.29	0.89 ± 0.34
CLNs	pFL	*6.1 ± 1.3	1.63 ± 0.24	*201.3 ± 55.3	*55.6 ± 14.9	*314.5 ± 66.6	1.51 ± 0.68	1.43 ± 0.41
	pORF	2.4 ± 0.2	1.13 ± 0.18	78.8 ± 13.6	18.8 ± 5.40	66.5 ± 28.8	0.72 ± 0.34	1.21 ± 0.47
Spleen	pFL	*4.8 ± 1.4	1.65 ± 0.47	*391.1 ± 68.9	*45.1 ± 10.5	*286.8 ± 53.4	*1.33 ± 0.50	1.57 ± 0.39
	pORF	1.9 ± 0.4	1.22 ± 0.39	53.3 ± 19.1	16.1 ± 6.20	57.7 ± 15.1	0.89 ± 0.42	1.40 ± 0.24

^aThe CD4⁺ T cells (4 × 10⁶ cells/ml) from lungs, CLNs, and spleen were isolated 7 days after the last immunization with rPspA (5 μg) and pFL or pORF as a mucosal adjuvant and cultured with T cell depleted feeder cells (8 × 10⁶ cells/ml). Values are presented as means ± SEM of data from 30 mice for each group and a total of three experiments. *, P < 0.05 when compared with mice given rPspA plus pORF.

^bProliferative responses of CD4⁺ T cells from mice nasally immunized with rPspA plus pFL or pORF as a mucosal adjuvant were represented as the stimulation index by measuring counts per minute (cpm) of wells with or without rPspA (control). The levels of [³H]TdR incorporation for each control well were between 500 and 1,000 cpm. The results show the individual values from these separate experiments of 30 mice per experimental group.

^cThe culture supernatant were harvested after 48 h of incubation and subjected to the respective cytokine ELISAs.

Protection against S. pneumoniae infection by nasal vaccination with rPspA plus pFL.

In order to determine the functional properties of nasal vaccination with rPspA plus pFL, mice were challenged with S. pneumoniae strain WU2 (1.8 × 10⁷ CFU/20 μl) 1 week after the last vaccination. When the bacterial densities in the lungs, NWs, and blood were examined 48 h after nasal challenge, mice given nasal rPspA plus pFL showed significantly lower bacterial density. Conversely, lungs, NWs, and blood of mice given rPspA plus pORF contained high numbers of S. pneumoniae bacteria (Fig. 5). These results show that nasal rPspA plus pFL provides effective protection against S. pneumoniae infection at the lung and nasal mucosa.

Fig. 5.

Comparison of protective effects against S. pneumoniae infection with nasal pFL-based rPspA vaccine. One week after the last immunization with rPspA plus pFL (closed circle) or pORF (open circle), mice were challenged with 1.8 × 10⁷ CFU of the WU2 strain. (A) Forty-eight hours after bacterial challenge, the lungs were removed aseptically and homogenized in 9 ml of sterile saline per gram of lung tissue for the culture. (B) NWs were harvested aseptically by flushing with 1 ml of PBS and cultured on agar medium. (C) Blood samples were plated on agar medium from the culture. The detection limit of bacterial cultures was 10² CFU/g. The values shown are the means ± SEM (n = 15). Each line represents the median log₁₀ CFU/mouse. *, P < 0.05, compared with mouse group given rPspA plus pORF.

DISCUSSION

In this study, we have investigated whether nasal pFL as a mucosal adjuvant elicits functional bacterial Ag (rPspA)-specific Ab responses for protection against S. pneumoniae infection. Our results clearly showed that nasal vaccination with rPspA plus pFL elicited DC-mediated Th2-type and IL-2 cytokine responses and subsequent anti-rPspA Abs for protection against pneumococcal infection at the pulmonary mucosa. Since a risk of central nervous system toxicity is one of the major issues for nasal vaccine development (6, 9, 29), we also examined pFL uptake and inflammatory cytokine synthesis in the nasal mucosa. Our results indicated that nasal pFL was taken up only by NALT and NP DCs, as well as nECs, but not by ON/E. Further, minimal levels of IL-1β, IL-6, and TNF-α production were induced in NWs of mice given pFL. Taken together, the current study is the first to show that nasal pFL is a safe mucosal adjuvant that effectively elicits bacterial Ag (PspA)-specific functional Ab responses that are potent for the prevention of pneumococcal pneumonia and bacteremia.

We recently showed that pFL as a nasal adjuvant elicited PspA-specific S-IgA Ab responses in the nasal cavity to prevent nasal carriage of S. pneumoniae (12). Although the recent study clearly indicated the potential of pFL as a nasal adjuvant for a pneumococcal vaccine, it still remained to be elucidated whether pFL can induce protection in the lower respiratory mucosa, including the lungs. In this regard, nasal pFL elicited functional rPspA-specific S-IgA Ab responses in the BALF and lungs when mice given nasal rPspA plus pFL were nasally challenged with a large amount (20 μl; 1.8 × 10⁷ CFU) of WU2 (invasive strain), allowing bacterial exposure to the lungs. Thus, mice given nasal rPspA plus pFL showed a significantly lower number of bacteria in the BALF after being challenged fatally with WU2 than did mice given empty plasmid as a nasal adjuvant. Based upon our previous studies and those of others (12, 42), we expected that nasal immunization with rPspA plus pFL induced Ag-specific functional S-IgA Ab responses in the nasal cavity. Indeed, a remarkable anti-rPspA S-IgA Ab induction and the inhibition of bacterial growth were seen in NWs after bacterial challenge. Thus, it is possible that an effective inhibition of nasopharyngeal bacterial colonization might lead to drastic reduction of bacterial growth in the lungs and prevention of subsequent bacteremia. In any case, the presence of anti-PspA IgA Abs in the lower and upper respiratory mucosa is the fundamental factor to prevent bacterial invasion of the hosts.

The FL protein is known as a synergistic hematopoietic growth factor that has emerged as a potential immunomodulator (8, 21, 38) and can expand DC populations and enhance antigen-presenting cell (APC) activity (13, 24). In addition, it was recently reported that percutaneous administration of the FL protein regulated migration and Ag uptake of lung DCs (34, 35). In this regard, our previous studies showed that nasal pFL increases the frequency of CD8⁺ DCs in various mucosal and systemic lymphoid tissues (11, 19). Our present study showed increased numbers of CD8⁺ DCs, which agrees with these previous findings even though a bacterial Ag was used as a component of the nasal vaccine. Since a recent study indicated that induction of CD8⁺ DCs promoted protection against respiratory infection (7), it is possible that induction of CD8⁺ DCs in the mucosal and systemic compartments contributes to S. pneumoniae clearance in the respiratory tract and blood. Further, increased frequencies of CD11b⁺ DCs were also seen in mice given nasal rPspA plus pFL. Recent reports showed that the interactions between CD4⁺ T cells and DCs play a key role in the induction of pulmonary immunity (1) and that DCs polarize initial CD4⁺ T cell activation toward Th2-type immune responses (33). Further, our previous and present studies showed that nasal pFL elicited CD8⁺ DC-mediated Th2-type responses. In this regard, it is possible that CD11b⁺ DCs play a role in the downregulation of Th2-prone cytokine responses for the maintenance of mucosal homeostasis. Indeed, it was suggested that FL treatment induced Th2-suppressive lung CD11b⁺ DCs (15, 35). Further, nasal application of FL-expressing adenovirus as a mucosal adjuvant preferentially expands CD11b⁺ DCs to produce a balanced Th1- and Th2-type cytokine response (32). The actual immunoregulatory functions of CD11b⁺ DCs induced by nasal pFL are currently being tested in our laboratory.

In summary, the present study shows that pFL as a nasal adjuvant induces enhanced PspA-specific immunity in the nasal-pulmonary mucosa via CD8⁺ and CD11b⁺ DC subset-mediated Th2-type cytokines responses. Importantly, nasal vaccination with rPspA plus pFL inhibits bacterial growth in the lungs and nasal cavities of mice in order to prevent the early phases of pneumococcal pneumonia without CNS toxicity or inflammation. These findings suggest that pFL is a safe nasal adjuvant for use in the future development of vaccines that can induce enhanced specific immunity against bacterial and viral Ags.