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Clinical and Vaccine Immunology, May 2009, p. 636-645, Vol. 16, No. 5
1071-412X/09/$08.00+0     doi:10.1128/CVI.00395-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization of Protective Mucosal and Systemic Immune Responses Elicited by Pneumococcal Surface Protein PspA and PspC Nasal Vaccines against a Respiratory Pneumococcal Challenge in Mice ^,

D. M. Ferreira,¹ M. Darrieux,¹ D. A. Silva,¹ L. C. C. Leite,¹ J. M. C. Ferreira Jr.,² P. L. Ho,¹ E. N. Miyaji,^1* and M. L. S. Oliveira^1*

Centro de Biotecnologia,¹ Laboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil²

Received 29 October 2008/ Returned for modification 3 February 2009/ Accepted 27 February 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Pneumococcal surface protein A (PspA) and PspC are virulence factors that are involved in the adhesion of Streptococcus pneumoniae to epithelial cells and/or evasion from the immune system. Here, the immune responses induced by mucosal vaccines composed of both antigens as recombinant proteins or delivered by Lactobacillus casei were evaluated. None of the PspC vaccines protected mice against an invasive challenge with pneumococcal strain ATCC 6303. On the other hand, protection was observed for immunization with vaccines composed of PspA from clade 5 (PspA5 or L. casei expressing PspA5) through the intranasal route. The protective response was distinguished by a Th1 profile with high levels of immunoglobulin G2a production, efficient complement deposition, release of proinflammatory cytokines, and infiltration of neutrophils. Intranasal immunization with PspA5 elicited the highest level of protection, characterized by increased levels of secretion of interleukin-17 and gamma interferon by lung and spleen cells, respectively, and low levels of tumor necrosis factor alpha in the respiratory tract.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Pneumococcal diseases kill more than 1 million children worldwide every year. The situation is worse in developing countries, where 90% of deaths occur. In Latin America, there are at least 1.6 million cases of pneumococcal disease every year, killing 18,000 children (53). While appropriate treatment, including the use of antibiotics and good nutrition, lowers the incidence of pneumococcal diseases, vaccines are the most efficacious way of preventing them. The existing pneumococcal conjugate vaccine dramatically reduces diseases, disabilities, and deaths, but elevated cost and protection restricted to included serotypes have prevented its implementation in large-scale immunization programs in developing countries. For these reasons, there is considerable interest in using conserved pneumococcal protein antigens as vaccines to provide cost-effective broad protection in all age groups. A number of leading candidates have been shown to elicit protection in mice (10, 51); among these antigens, two of the most promising candidates are pneumococcal surface protein A (PspA) and PspC.

An additional concern in the development of cost-effective vaccines against pneumococcal disease is the route of immunization. Human vaccines are traditionally administered intramuscularly by needle inoculation, which brings the risk of transmitting blood-borne pathogens such as human immunodeficiency virus and hepatitis viruses (20). Furthermore, the cost of equipment and well-trained personnel for delivering vaccines by parenteral routes is several times higher than the cost of the vaccines themselves. This aspect is extremely important for vaccine implementation in large-scale immunization programs for developing countries. Mucosal delivery of pediatric vaccines has become an explicit goal of the WHO (20). Immunization via mucosal surfaces would greatly increase the ease of vaccination and would be more readily acceptable than parenteral immunization in many populations. Therefore, the move from injection to mucosal application would be very positive from economical, logistical, and safety standpoints. Mucosal immune responses are also important for the prevention of many infectious diseases because they represent the first barrier from the hosts that pathogens must evade.

Research into the host immune response to pneumococcal diseases has focused primarily on the role of innate and adaptative humoral immune responses. However, in the last few years, attention has been drawn to cellular immune responses against Streptococcus pneumoniae, with interesting results. The majority of these studies analyzed cellular aspects of innate immunity and proposed that lymphocytes, neutrophils, and macrophages orchestrate effective immune responses without the presence of specific antibodies. In this context, proinflammatory cytokines promote an adequate milieu for pneumococcal clearance (22, 24, 25, 31, 34, 50, 54). A Th1-biased immune response has also been shown to be engaged in the resolution of pneumococcal infection in humans (21). Nevertheless, inflammatory cell influx into the lung and mucosal responses must be regulated to avoid exacerbated tissue injury. This is evidenced in recent studies of the role of T cells and/or anti-inflammatory cytokines, such as interleukin-10 (IL-10), in pneumococcal infection (26, 42, 55).

Protective immune responses against invasive pneumococcal disease and colonization were shown using pneumococcal whole-cell vaccines (28, 46) or recombinant proteins as mucosal vaccines (2, 6, 7, 9, 40). In recent approaches, lactic acid bacteria (3, 11, 18, 37), which are able to activate and modulate the innate immune system (35, 42), were used for pneumococcal antigen presentation, with promising results.

Very few works compared pulmonary and systemic immune responses induced by pneumococcal antigens using parenteral and mucosal immunizations (13). The present study aims at investigating local and systemic cellular and humoral immune responses required for protection against invasive intranasal (i.n.) challenge with S. pneumoniae strain ATCC 6303 using PspA and PspC antigens administered by both routes, without the use of adjuvants, or presented by Lactobacillus casei through the nasal route.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bacterial strains and growth conditions. Lactococcus lactis cultures were grown in M17 medium (Difco); L. casei CECT5275 cultures were grown in Man, Rogosa, and Sharpe medium (Difco); and S. pneumoniae ATCC 6303 cultures (serotype 3, PspA clade 5) were grown in Todd-Hewitt broth (Sigma) supplemented with 0.5% yeast extract (THY). Bacteria were grown at 37°C without shaking. Pneumococcal strain ATCC 6303 was always plated in blood agar and grown overnight at 37°C before inoculation in THY. All bacterial stocks were maintained at –80°C in their respective media containing 20% glycerol.

Recombinant proteins. Expression and purification of the N-terminal fragment of PspA from clade 5 (from strain 122/02, serotype 23F) were performed as previously described (14). The fragment encoding the PspC N-terminal region was amplified from S. pneumoniae strain 491/00 (Instituto Adolfo Lutz, São Paulo, Brazil) using forward primer 5'-TAGGGATCCCATGCGACAGAGAACGAGA-3' and reverse primer 5'-CTGCAGTTATTGTGGTTGTTCAGC-3'. The gene product was cloned into a pGEMT-Easy vector (Promega), and the sequence was confirmed by DNA sequencing. The fragment was subcloned into linearized vector pAE (43) and used to transform Escherichia coli BL21(DE3) SI competent cells (Invitrogen). Protein expression was induced in mid-exponential-phase cultures by the addition of 300 mM NaCl. The recombinant proteins bearing N-terminal histidine tags were purified from the soluble fraction by affinity chromatography using Ni²⁺-charged resin (HisTrap HP; GE Healthcare). Elution was carried out with 250 mM imidazole. The purified fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, dialyzed against 10 mM Tris-HCl (pH 8.0)-20 mM NaCl-0.1% glycine, and stored at –20°C.

Cloning and recombinant procedures in lactic acid bacteria. Vector pT1NX (11) was used for the constitutive intracellular expression of the N-terminal region of PspA5 and PspC in L. casei. The following primers were used for amplification: PspAlacF (5'-ATGCATCGATATCAGAAGAAGCTCCCGTAGCT-3') and PspAlacR (5'-GGATCCTTAAGATCTTTTTGGTGCAGGAGCAGCTGG-3') for the amplification of pspA5 and PspClacF (5'-ACCGGTGATATCCCATGCGACAGAGAACGAG-3') and PspClacR (5'-GGATCCTTAAGATCTTTGTGGTTGTTCAGC-3') for the amplification of pspC. After sequencing confirmation, the fragments were cloned into vector pT1NX, and ligation products were used to transform competent L. lactis cells as previously described (37). Plasmids isolated from L. lactis were then used for the electroporation of L. casei (37). L. lactis and L. casei transformants were selected by plating onto the respective medium containing 1.8% agar and 5 µg/ml of erythromycin. Protein expression was confirmed by Western blotting of L. casei lysates using specific antibodies.

Immunization of mice. Five- to seven-week-old female C57BL/6 mice from the Central Animal Facility of Butantan Institute were supplied with food and water ad libitum. Animal experimental protocols were approved by Use Ethics Committee of Instituto Butantan (São Paulo, Brazil). Groups of 4 to 10 animals were anesthetized through the intraperitoneal (i.p.) route with 200 µl of a mixture of 0.5% xylazine and 0.2% ketamine and inoculated i.n. with 6 doses (5 µg in 10 µl) on days 0, 3, 14, 17, 28, and 31 or subcutaneously (s.c.) with 3 doses on days 0, 14, and 28 (5 µg in 100 µl) of PspC or PspA5 previously treated with Triton X-114 to remove lipopolysaccharide as described previously (1) and without the use of adjuvants. L. casei cells expressing PspC or PspA5 or carrying the empty vector were grown until stationary phase (optical density at 550 nm [OD₅₅₀] of 2), collected by centrifugation, washed with saline, and then suspended to 10⁹ viable cells in 10 µl. The cell suspension was inoculated i.n. on days 0, 1, 14, 15, 28, and 29.

Detection of anti-PspC- and anti-PspA-specific antibodies through ELISA. Mice were bled through the retroorbital plexus 15 days after the last immunization. Vaginal washes were collected from days 15 to 19 after the last immunization by gentle pipetting of 25 µl of saline twice, and samples were pooled for each animal. Antibody levels in serum, vaginal washes, and bronchoalveolar lavage fluid (BALF) (collected as described below) were evaluated by enzyme-linked immunosorbent assay (ELISA) in plates coated with PspC or PspA5. The assay was performed using goat anti-mouse immunoglobulin G [IgG], IgA, IgG1, or IgG2a and rabbit anti-goat antibody conjugated with horseradish peroxidase (Southern Biotech). Differences in antibody titers were analyzed by the Mann-Whitney U test, and a P value of 0.05 was considered to be significantly different. Titers were defined as the last dilution in which absorbances at 492 nm reached 0.1.

Antibody binding and complement deposition assays. Frozen stocks of S. pneumoniae ATCC 6303 were plated onto blood agar overnight and then grown in THY to an OD₆₀₀ of 0.4 to 0.5 (10⁸ CFU/ml) and harvested by centrifugation. Bacteria were washed, resuspended in phosphate-buffered saline (PBS), and incubated with 10% of pooled sera during 30 min at 37°C. Samples were washed once with PBS before incubation with fluorescein isothiocyanate-conjugated anti-mouse IgG (Sigma) for 30 min on ice. For complement deposition assays, sera were previously heated at 56°C for 30 min and incubated with bacteria at a concentration of 25% at 37°C for 30 min. Samples were washed once with PBS and incubated with 10% normal mouse serum as the source of complement in Gelatin Veronal buffer (Sigma) at 37°C for 30 min. After washing, samples were incubated with fluorescein isothiocyanate-conjugated anti-mouse C3 IgG (MP Biomedicals) in PBS for 30 min on ice. Samples were fixed with 2% formaldehyde after two washing steps and stored at 4°C. Flow cytometry analysis was conducted using a FACSCalibur apparatus (Becton Dickinson), and 10,000 gated events were recorded. The median of fluorescent bacteria was used to compare the groups.

i.n. challenge. i.n. challenge was performed to monitor mouse survival and to analyze immune cell responses, since in previous work from our group, such responses were not detected in nonchallenged immunized mice (our unpublished data). S. pneumoniae ATCC 6303 cells were grown in THY medium until the OD₆₀₀ reached 0.4, aliquoted, and kept frozen at –80°C. A suspension containing 10⁵ bacteria in 30 µl was inoculated into one nostril of mice previously anesthetized through the i.p. route with 200 µl of a mixture of 0.5% xylazine and 0.2% ketamine 21 days after the last immunization. Survival was monitored for 10 days. Differences in survival rates were analyzed by Fisher's exact test, and differences in survival time were measured by Kaplan-Meier survival curve analysis. In both cases, a P value of 0.05 was considered to be significantly different.

Collection of BALF and cytospin. Mice were sacrificed 13 h after i.n. pneumococcal challenge by injection of a lethal dose of urethane (15 mg per 10 g of body weight) to collect the spleen, lung, and BALF samples. A catheter was inserted into the trachea of the mice, and lungs were rinsed with 0.5 ml of sterile PBS, followed by an additional rinse with 1 ml of PBS. The fluids from both rinses were pooled. Cells obtained from the pooled fluid were washed and resuspended in PBS and counted, and 4 x 10⁴ cells were spun onto glass slides (4 min at 1,300 rpm) by the use of a cytocentrifuge (StatSpin Cytofuge). Cytospin slides were stained, and 100 cells were differentially counted to analyze the percentage of infiltration of each cell type. Fluid samples were stored at –80°C for subsequent analyses.

Detection of antigen-specific cytokines. Lung tissue was dissected into small pieces and digested with a collagenase-DNase solution (collagenase type IV-DNase-150, 50 units/ml; Sigma-Aldrich). Single-cell suspensions of spleen were obtained as described previously (17). Viable cell counts were determined by trypan blue exclusion. Lung and spleen cells from each group were pooled, and cells secreting gamma interferon (IFN-) were detected using an enzyme-linked immunospot (ELISPOT) set (BD Biosciences) as described previously (17) using dilutions from 5 x 10⁶ to 1 x 10⁷ cells/ml and stimulation with recombinant proteins (5 µg/ml) for 20 h. The average number of spot-forming cells (SFCs) was calculated from duplicate wells, subtracting nonstimulated wells. Detection of IL-17, tumor necrosis factor alpha (TNF-), and IL-5 secretion in the supernatants of spleen and lung cell cultures stimulated for 72 h with recombinant proteins (5 µg/ml) was performed by use of sandwich ELISA (BD Biosciences) using 1 x 10⁷ cells/ml.

Nucleotide sequence accession number. The nucleotide sequence for the PspC gene fragment was deposited in GenBank under accession no. EF424119.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Infiltration of cells after i.n. pneumococcal challenge in PspC-immunized mice. Mice were i.n. immunized with the PspC protein without the use of an adjuvant or L. casei cells expressing this antigen. Animals immunized with L. casei cells carrying the empty vector (L. casei) or nonimmunized animals were used as control groups. An additional group received PspC s.c. Animals were bled 15 days after the last immunization, and antibodies in sera and vaginal washes were analyzed through ELISA. Specific anti-PspC IgGs were detected only in sera from s.c.-immunized animals (IgG titer of 1:800). For the other groups, no reactivity was observed even at the lowest dilution tested (1:20). In addition, anti-PspC IgA was not observed in vaginal washes collected from immunized animals (the minimal dilution tested was 1:4). Twenty-one days after the last immunization, all groups were challenged with strain ATCC 6303 through the i.n. route, and 13 h after challenge, BALF and tissue samples were collected. The time point of 13 h was chosen because at this time, the animals do not yet show any sign of the disease, and there is no detectable CFU in blood. In addition, no differences in lung CFU counts among all groups were observed at this point (data not shown). Even 13 h after challenge, anti-PspC antibodies were not detected in BALF samples by ELISA. Analysis of cells recovered from BALF (Fig. 1) showed a significant infiltration of neutrophils in L. casei PspC-immunized animals compared with the L. casei control group (P = 0.0004). Nasal immunization with PspC also induced an augmentation in neutrophil infiltration compared with neutrophil infiltration in nonimmunized animals, but this difference was not statistically significant. No difference was observed in the percentages of neutrophils recovered from mice s.c. immunized with PspC compared with nonimmunized animals.

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FIG. 1. BALF cell counts. Slides were prepared with 4 x 104 cells from BALF. Percentages of infiltrated cells are expressed as means of data from four to five mice per group. # represents a significant difference in neutrophil infiltration between the group immunized with L. casei (L.c.) and the group immunized with L. casei PspC (P = 0.0004 by Mann-Whitney U test). Non, nonimmunized group.

IFN- and IL-17 secretion by lung and spleen cells in PspC-immunized mice. After collection of BALF, lung and spleen were excised from mice. Secretion of IFN- and IL-17 from pooled cells of each group was detected through ELISPOT assay and sandwich ELISA, respectively. As observed in Fig. 2A, nasal immunizations with L. casei PspC and PspC were able to induce an increase in the number of lung cells secreting IFN- compared with control groups or even with the group immunized s.c. with PspC. This effect was more pronounced for L. casei PspC than for PspC nasal immunization. IFN- secretion from spleen cells was also analyzed, and PspC immunization was able to induce an increase in numbers of SFCs when administered through the s.c. route. Interestingly, the L. casei PspC-immunized group showed the same level of IFN--secreting cells as the group immunized s.c. with PspC, whereas no induction of IFN- secretion by spleen cells was observed in the group i.n. immunized with PspC (Fig. 2B). As for IL-17, all immunized groups displayed elevated levels of secretion of this cytokine in lung cell culture supernatants. The most pronounced secretion was observed in the group immunized i.n. with PspC that presented an eightfold increase in the concentration of this cytokine (Fig. 2C). On the other hand, this immunization induced fourfold-less IL-17 secretion than L. casei PspC immunization in spleen cells, whereas subcutaneous immunization with PspC did not produce any increase at all (Fig. 2D).

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FIG. 2. Cellular immune response in lung (A and C) and spleen (B and D) induced by immunization with PspC antigen after i.n. challenge with strain ATCC 6303. Spleen and lung cells were isolated from immunized mice 13 h after i.n. challenge and incubated with PspC in ELISPOT plates previously coated with anti-IFN-. SFCs of IFN- were detected through ELISPOT, and the average number of spots in duplicate wells was calculated by subtracting nonstimulated wells and considered as the number of SFCs/106 cultured cells (A and B). Cells were also incubated with PspC, and IL-17 secretion in the supernatants was detected through sandwich ELISA (C and D). Results are representative of two independent experiments. L.c., L. casei; Non, nonimmunized group.

Serum and mucosal anti-PspA antibody responses. The levels of anti-PspA antibodies were measured by ELISA. As shown in Fig. 3A, i.n. immunization of mice with PspA5 elicited significantly higher IgG responses than in nonimmunized mice (P = 0.0001). L. casei PspA5 immunization also led to a significant increase in levels of anti-PspA antibodies compared with those found with L. casei immunization (P = 0.002). s.c. immunization with PspA5 induced an elevated level of antibody production compared with that of nonimmunized animals (P = 0.001), but the level was slightly lower than the one observed for mice immunized i.n. with PspA5. i.n. immunization induced intermediate anti-PspA5 IgG1/IgG2a ratios in protein groups (IgG1/IgG2a ratio of 91) and lower ratios when the antigens were delivered by L. casei (IgG1/IgG2a ratio of 0.1) (Fig. 3B). The highest ratio was observed in the group immunized with PspA5 s.c. (IgG1/IgG2a ratio of 358). Specific anti-PspA secreted IgA (sIgA) was detected only in vaginal washes obtained from mice i.n. immunized with PspA5 (Fig. 3C). These antibodies were not detected in samples from the other groups, even at a 1:4 dilution.

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FIG. 3. Evaluation of anti-PspA antibodies in mice immunized with PspA5 vaccines. Two weeks after the last immunization, anti-PspA IgG (A), IgG1, and IgG2a (B) in sera and IgA in vaginal washes (C) were detected through ELISA. Log10 values of antibody titers are shown, and IgG1/IgG2a titer ratios are indicated above the bars (B). Results are representative of two experiments. Asterisks represent significant differences from the indicated control group (*, P = 0.002; **, P = 0.001; ***, P = 0.0001 [Mann-Whitney U test]). L.c., L. casei; Non, nonimmunized group.

Binding of anti-PspA antibodies and complement deposition onto ATCC 6303. In order to investigate whether anti-PspA antibodies detected by ELISA would be able to bind to the surface of intact pneumococci and mediate C3 deposition, sera from mice immunized i.n. or s.c. with PspA5 or immunized i.n. with L. casei PspA5 were incubated with strain ATCC 6303 in an antibody binding assay. Sera from nonimmunized mice or mice immunized with L. casei were used as controls. As shown in Fig. 4A, we observed that L. casei PspA5 immunization elicited antibodies that showed the highest binding capacity, which was especially surprising compared with s.c. immunization with PspA5. Furthermore, when the ability to mediate C3 deposition onto the pneumococcal surface was analyzed (Fig. 4B), sera from animals i.n. inoculated with L. casei PspA5 or PspA5 elicited a more pronounced increase in C3 deposition than did sera from mice s.c. immunized with PspA5. It is important that PspA5 immunization through the s.c. route, without adjuvant, elicited larger amounts of specific PspA5 antibodies than did the L. casei PspA5 group (Fig. 3A). However, these Igs did not display the same efficacy in binding onto the pneumococcal surface and enhancing complement deposition.

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FIG. 4. Binding of anti-PspA5 antibodies and complement deposition onto the pneumococcal surface. Sera from mice i.n. immunized with PspA5 (dotted lines) or L. casei PspA5 (solid heavy lines) or s.c. immunized with PspA5 (solid thin lines) were tested for the ability to bind to the pneumococcal surface (A) and to mediate C3 deposition (B). S. pneumoniae ATCC 6303 was incubated with 10% (A) or 25% (B) serum from each group serum. Sera from nonimmunized (Non) animals (gray areas) and animals immunized with L. casei (L.c.) (dashed lines) were used as controls. The median fluorescence of the bacteria is shown for each sample. Data are representative of two independent experiments. FITC, fluorescein isothiocyanate.

Enhancement of infiltration of neutrophils and presence of anti-PspA5 antibodies in BALF after i.n. pneumococcal challenge. BALF cell samples were recovered from animals immunized with PspA5 vaccines 13 h after a respiratory challenge with strain ATCC 6303. A significant increase in the level of infiltration of neutrophils was observed in mice immunized with L. casei PspA5 compared with mice immunized with L. casei (P = 0.005) (Fig. 5). BALF recovered from mice immunized i.n. with PspA5 also demonstrated an increase in levels of these cells compared with nonimmunized mice (P = 0.001). Further investigation showed that only mice immunized i.n. with PspA5 presented anti-PspA5 sIgA (mean log of antibody titer of 1.12 ± 0.51) in BALF recovered after pneumococcal challenge (data not shown).

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FIG. 5. BALF cell counts. Slides were prepared with 4 x 104 cells from BALF. Percentages of infiltrated cells are expressed as means of data from four to five mice per group. An * represents a significant difference in neutrophil infiltration between mice immunized i.n. with PspA5 and nonimmunized mice (Non). ** represents a significant difference in neutrophil infiltration from mice immunized with L. casei PspA5 and L. casei (L.c.) (*, P = 0.001; **, P = 0.005 [Mann-Whitney U test]).

Cytokine secretion by lung and spleen cells in PspA5-immunized mice. Analyzes of the cellular immune response showed that L. casei PspA5 immunization led to an intense increase in the number of lung cells secreting IFN- (fourfold increase) compared with other groups, including mice immunized i.n. with PspA5 (Fig. 6A). In spleen cells, the highest level of IFN- secretion was observed in the group immunized with PspA5 s.c., followed by PspA5 i.n. immunization (Fig. 6B). Conversely, IL-17 secretion was augmented in lung cells obtained from the groups immunized with PspA5 s.c. and PspA5 i.n. (Fig. 6C). This last group presented the most pronounced increase in levels of IL-17 secretion in the supernatant of lung and spleen cells (Fig. 6D). The level of IL-5, a Th2 cytokine, was also measured, and IL-5 was detected only in the group immunized s.c. with PspA5 (Fig. 6E). Additionally, the group immunized with PspA5 i.n. displayed the lowest level of secretion of the proinflammatory cytokine TNF- by lung cells 13 h after pneumococcal challenge (Fig. 6F), indicating a controlled inflammatory response.

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FIG. 6. Cellular immune response in lung (A, C, E, and F) and spleen (B and D) induced by immunization with PspA5 antigen after i.n. challenge with strain ATCC 6303. Spleen and lung cells were isolated from immunized mice 13 h after i.n. challenge and incubated with PspA5 in ELISPOT plates previously coated with anti-IFN-. SFCs of IFN- were detected through ELISPOT assay, and the average number of spots in duplicate wells was calculated by subtracting nonstimulated wells and considered as the number of SFCs/106 cultured cells (A and B). Cells were also incubated with PspA5, and IL-17 (C and D), IL-5 (E), and TNF- (F) secretion in the supernatants was detected through sandwich ELISA. Results are representative of two independent experiments. L.c., L. casei; Non, nonimmunized group.

Evaluation of protection conferred by PspC and PspA vaccines against invasive i.n. challenge. Mice immunized with the different PspC- and PspA-based vaccines were challenged 21 days after the last immunization with strain ATCC 6303 through the i.n. route and were sacrificed 13 h later for determinations of CFU in lungs or were monitored during 10 days for observation of survival. No significant changes in CFU counts in the lungs were observed among all the groups 13 h after challenge (data not shown). By analyzing PspC-based vaccines, we observed that despite the cellular immune responses elicited, no significant survival was observed in mice immunized with any formulation or by any administration route (Table 1). Sequence comparison of the N-terminal region of the PspC molecule used in our vaccines and ATCC 6303 PspC showed 47% amino acid conservation (identical plus conserved residues). Vaccines composed of PspC molecules with higher degrees of conservation remain to be tested in this model. On the other hand, as shown in Table 2, i.n. PspA5 and L. casei PspA5 immunizations were able to confer significant protection against this invasive pneumococcal challenge compared with nonimmunized mice (60% for mice immunized with PspA5 i.n. and 40% for mice immunized with L. casei PspA5 i.n.) (P = 0.002 and P = 0.02, respectively, by Fisher's exact test). Through this type of analysis, s.c. administration of PspA5 was not able to confer protection against this challenge model. Kaplan-Meier analysis confirmed significant protection in mean survival times for animals i.n. immunized with PspA5 (P = 0.0006) and L. casei PspA5 (P = 0.005) compared with nonimmunized mice. An increase in mean survival time was also observed in animals s.c. immunized with PspA5 (P = 0.04) (Fig. 7). The L. casei control group did not elicit significant protection in this pneumococcal challenge model neither in total survival rate (P = 0.21) nor in mean survival time analysis (P = 0.15).

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TABLE 1. Survival of mice after i.n. challenge with S. pneumoniae ATCC 6303

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TABLE 2. Survival of mice after i.n. challenge with S. pneumoniae ATCC 6303

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FIG. 7. Mean survival time of mice after i.n. challenge. Immunized mice were challenged with 105 CFU of pneumococcal strain ATCC 6303 through the i.n. route, and survival was monitored for 10 days. Results were evaluated by Kaplan-Meier survival curve analysis. A P value of 0.05 was considered to be significantly different (*). L.c., L. casei.

Histological analysis of lung tissue 13 h after challenge revealed an increase in cell infiltration in mice immunized i.n. with PspA5, L. casei, or L. casei PspA5 compared with nonimmunized mice or mice immunized s.c. with PspA5. However, no inflammatory injuries were observed in any group at this point (see Fig. S1 in the supplemental material).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The precise mechanisms of the resolution of S. pneumoniae lung infection conferred by effective mucosal vaccines have not yet been elucidated. In the present study, we used two of the most promising pneumococcal vaccine candidates, PspC and PspA, through different routes of immunization (mucosal and parenteral) and also used the lactic acid bacterium L. casei as a delivery system of these antigens in order to evaluate the induction of pulmonary and systemic immune responses and their protective potential against an invasive challenge with S. pneumoniae in mice.

The investigation of PspC-based vaccines revealed that the group vaccinated with L. casei expressing this antigen was the only group that presented a significant enhancement in the infiltration of neutrophils in BALF compared with the control group inoculated with L. casei. As for the humoral response, antibodies against PspC were observed only in sera from mice s.c. immunized with the recombinant protein. The better efficacy of the s.c. route over the mucosal route of immunization for the induction of anti-PspC IgG in sera may be a consequence of differences in antigen capture and presentation as well as protein stability in each environment, favoring s.c. immunization. None of the formulations were able to elicit mucosal antibodies under the conditions tested. Immunization with recombinant PspC through different routes has been shown to induce specific antibodies and to confer protection against different pneumococcal challenge models (7, 12, 36). The absence of anti-PspC antibodies in mice immunized with our PspC protein-based vaccines maybe explained by the fact that we have removed residual lipopolysaccharides resultant from E. coli purification and tested them in the absence of adjuvants. It is also important that the experiments described in the present work were performed using C57BL/6 mice, whereas previous works used BALB/c mice. It is known that the susceptibilities of these two mouse strains to pneumococcal infection are different and may also have contributed to the differences observed (19).

The cellular immune response was characterized by the secretion of IFN- and IL-17 by lung and/or spleen cells. Despite the detection of antigen-specific cellular immune responses, protection was not observed in PspC-immunized mice. However, further studies to analyze the protective capacity of vaccines composed of PspC molecules displaying a higher degree of conservation with the PspC-expressed ATCC 6303 must be considered.

In our hands, PspA5 was shown to be more immunogenic than PspC. Antibodies elicited by all PspA5 formulations were able to bind to strain ATCC 6303. Even with lower levels of IgG induced by L. casei PspA5, serum obtained from this group showed superior binding compared with those of sera from other groups. These results may be explained by the fact that the antigen expressed and delivered by L. casei may have a more appropriate folding, resulting in antibodies that recognize epitopes present in the PspA molecule attached to the pneumococcal surface.

Nasal PspA5 vaccines displayed higher levels of IgG2a than did the s.c. vaccine, which was reflected in more balanced IgG1/IgG2a ratios. Nevertheless, the L. casei PspA5 group was the only group that displayed larger amounts of IgG2a than IgG1. These isotyping results are in accordance with previous work with lactic acid bacteria showing that L. lactis administration through the i.n. route induced a Th1-biased immune response with higher levels of IgG2a production, while its administration though the i.p. route led to the production of elevated amounts of IgG1 (45). It has also been reported that lactobacilli may facilitate the polarization of the naive immune system by skewing it away from Th2 responses and toward Th1 immune responses (35).

When evaluating complement deposition, we observed that the groups immunized i.n. with L. casei PspA5 and PspA5 presented the greatest ability in mediating complement deposition onto the pneumococcal surface. Interestingly, complement deposition observed with sera from L. casei PspA5-immunized mice was similar to that observed for sera from mice immunized i.n. with PspA5 despite the smaller amounts of antibody elicited. These results can be explained by the fact that the lactobacillus vaccine elicited predominantly IgG2a, in contrast with the other groups. In a previous work by our group, we proposed that since anti-PspA IgG2a would be the isotype with the greatest capacity to mediate complement deposition onto the pneumococcal surface, large amounts of IgG1 (in this case elicited by s.c. immunizations) could compromise complement deposition by impairing the binding of IgG2a antibodies (17). Arulanandam and colleagues have also shown that Th1 immune responses, as the one elicited by PspA using IL-12 as an adjuvant through the i.n. route, led to the production of large amounts of IgG2a and IgA and correlated this increase with protection against lung infection. Blocking these Igs with specific proteins significantly inhibited pneumococcal uptake by phagocytic cells (2).

i.n. immunization with PspA5 also induced specific sIgA antibodies detected in vaginal washes 2 weeks after the last immunization and in BALF samples 13 h after challenge. This finding correlated with better protection in our model. The role of sIgA against colonization by S. pneumoniae in nasal mucosa is controversial. While some studies demonstrated that protection can occur in the absence of B cells (30, 31), a crucial role for sIgA was demonstrated in a model using pIgR^–/– and IgA^–/– mice (49). Still, it is well known that sIgA exerts protection by noninflammatory mechanisms such as the inhibition of bacterial adhesion or opsonization not mediated by complement (39). Such a response may therefore contribute to the survival of mice.

The highly encapsulated strain ATCC 6303 (serotype 3) is extremely virulent to mice in the i.n. challenge used in this study. In vitro studies have shown that neither human nor rat polymorphonuclear cells are able to phagocytose this strain (8, 16). Even with this high virulence, in this study, we showed a significant protection of mice i.n. immunized with PspA5 (60%) or with L. casei PspA5 (40%) compared with nonimmunized animals. It is important that the pspA5 fragment used in this study was amplified from a Brazilian pneumococcal isolate (strain 122/02, serotype 23F) (14) and not from the same strain used for the challenge. Using the s.c. route, protection was observed only by mean survival time analysis, showing that i.n. immunization was important to elicit an adequate immune response required in this challenge model.

Elevated levels of IFN- and low levels of IL-17 secretion by lung cells were observed after immunization with L. casei PspA5. The opposite (high levels of IL-17 and low levels of IFN-) was observed for i.n. immunization with PspA5. The protective role of IL-17 in a mouse model of pneumococcal colonization was very well characterized by the works of Malley and collaborators. In those studies, the authors elegantly showed the role of IL-17-expressing CD4⁺ T cells in the protection conferred by a killed whole-cell vaccine. However, all the correlations determined to date are related to protection against the pneumococcal nasopharyngeal colonization model (27, 29, 52). IL-17 acts on the recruitment of neutrophils to sites of infection and is also involved in pulmonary host defenses against various pathogens (4, 5, 41). Nevertheless, we have no knowledge of data on IL-17 expression by lung cells in mouse models of pneumococcal infection. In our model, increases in levels of IL-17 secretion and neutrophil infiltration after challenge were observed for i.n. immunization with PspA5, correlating with the highest protection level. In L. casei PspA5-immunized mice, neutrophil infiltration was also observed, but no IL-17 secretion was observed 13 h after challenge, suggesting different mechanisms for protection elicited by the two vaccines.

It was recently reported that i.n. IL-12 administration in naive mice led to IFN- production by NK cells and TNF- production by macrophages in the lung, increasing neutrophil recruitment (50). Nonrecombinant lactobacilli are known to induce high levels of IL-12 release, inhibiting Th2 cytokine responses (IL-4 and IL-5) (35). Although a Th1-biased immune response seems to be a key component of the host defense against invading pathogens such as pneumococcus, this polarized immune response has also been implicated in inflammatory disorders (32, 33) and may cause lung injury (48). Secretion of IFN- by lung cells and protection against pneumonia were described previously (50, 54). On the other hand, in some cases, secretion of this cytokine seems to be detrimental (44, 47). In a mouse model of pneumonia, it has been shown that elevated levels IFN- secreted by NK cells present in lungs of scid mice are unfavorable for recovering from infection (23). Accordingly, the high levels of IFN- released by lung cells observed in the L. casei PspA5-immunized group may be prejudicial, resulting in only 40% protection. Other aspects of such polarized immune response are beneficial, such as the large amounts of IgG2a, leading to enhancement in complement deposition.

Previous studies reported that the release of TNF- early in infection would be beneficial but that its levels have to be controlled (22). Protection against lung injuries caused by S. pneumoniae infection after oral administration of nonrecombinant L. casei was related to a balanced induction of the proinflammatory cytokine TNF- and the anti-inflammatory IL-10, leading to a rapid increase in the infiltration of neutrophils with subsequent control of the inflammatory response in the lung of treated animals (42).

Although no histological signs of inflammatory injuries were observed 13 h after challenge, a possible deleterious effect, in advanced stages, of the intense release of TNF- observed in control groups as well as in groups i.n. immunized with L. casei PspA5 or s.c. immunized with PspA5 cannot be discarded. The group of mice i.n. immunized with PspA5 was the only group with low levels of TNF- release by lung cells 13 h after challenge, and this was exactly the group that presented the best protection.

Protection using lactic acid bacteria as an antigen delivery system was previously reported with L. lactis or L. casei cells expressing PspA against respiratory or i.p. challenge with different pneumococcal strains (11, 18). However, those previous works did not clarify the mechanisms involved in protection against S. pneumoniae. The present work is the first to aim at characterizing both humoral and cellular immune responses induced locally (lung) and systemically (spleen) by the administration of a pneumococcal antigen through a lactic acid bacterium delivery system. Furthermore, quantitative and qualitative features of mucosal and s.c. immunizations have been identified. Differences between the two routes of administration using two promising protein vaccine candidates were evaluated. The bias toward the Th1 immune response obtained by i.n. immunization with PspA5 is of particular interest since IFN- as well as IgG2a are critical in defense mechanisms against pneumococci. A concern about the administration of antigens through the i.n. route is the need for a safe and good adjuvant (38). Different tests showed the difficulty in maintaining adjuvanticity while reducing toxic effects of an adjuvant molecule (15). In this work, we showed that PspA5 is highly immunogenic and is able to induce protective humoral and cellular immune responses through i.n. administration without the use of adjuvant. We thus propose that the protection against this invasive challenge is likely due to both complement deposition induced by specific anti-PspA5 IgG2a and elevated pulmonary immunity with secretion of proinflammatory cytokines. Nevertheless, control of TNF- cytokine release is important to increase protection.

 
We are deeply grateful to Ana Paula Christ for help with the implementation of BALF collection protocols and Vania Gomes de Mattaria for the animal facilities coordination.

This work was supported by CAPES, CNPq, FAPESP, Fundação Butantan, and Millenium Institute-Gene Therapy Network (MCT-CNPq).

 
* Corresponding author. Mailing address: Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, Brazil. Phone: 55-11-3726-7222, ext. 2244. Fax: 55-11-3726-1505. E-mail for M. L. S. Oliveira: mloliveira{at}butantan.gov.br. E-mail for E. N. Miyaji: enmiyaji{at}butantan.gov.br

Published ahead of print on 11 March 2009.

Supplemental material for this article may be found at http://cvi.asm.org/.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Clinical and Vaccine Immunology, May 2009, p. 636-645, Vol. 16, No. 5
1071-412X/09/$08.00+0     doi:10.1128/CVI.00395-08
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