characterization of protective mucosal and systemic ...(20). immunization via mucosal surfaces would...

CLINICAL AND VACCINE IMMUNOLOGY, May 2009, p. 636–645 Vol. 16, No. 51556-6811/09/$08.00�0 doi:10.1128/CVI.00395-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization of Protective Mucosal and Systemic Immune ResponsesElicited 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. Oliveira1*Centro de Biotecnologia1 and Laboratorio de Imunoquímica,2 Instituto Butantan, Sao Paulo, Brazil

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

Pneumococcal surface protein A (PspA) and PspC are virulence factors that are involved in the adhesion ofStreptococcus pneumoniae to epithelial cells and/or evasion from the immune system. Here, the immuneresponses induced by mucosal vaccines composed of both antigens as recombinant proteins or delivered byLactobacillus casei were evaluated. None of the PspC vaccines protected mice against an invasive challenge withpneumococcal strain ATCC 6303. On the other hand, protection was observed for immunization with vaccinescomposed of PspA from clade 5 (PspA5 or L. casei expressing PspA5) through the intranasal route. Theprotective 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. Intra-nasal immunization with PspA5 elicited the highest level of protection, characterized by increased levels ofsecretion of interleukin-17 and gamma interferon by lung and spleen cells, respectively, and low levels of tumornecrosis factor alpha in the respiratory tract.

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

An additional concern in the development of cost-effectivevaccines against pneumococcal disease is the route of immu-nization. Human vaccines are traditionally administered intra-muscularly by needle inoculation, which brings the risk oftransmitting blood-borne pathogens such as human immuno-deficiency virus and hepatitis viruses (20). Furthermore, thecost of equipment and well-trained personnel for delivering

vaccines by parenteral routes is several times higher than thecost of the vaccines themselves. This aspect is extremely im-portant for vaccine implementation in large-scale immuniza-tion programs for developing countries. Mucosal delivery ofpediatric vaccines has become an explicit goal of the WHO(20). Immunization via mucosal surfaces would greatly in-crease the ease of vaccination and would be more readilyacceptable than parenteral immunization in many populations.Therefore, the move from injection to mucosal applicationwould be very positive from economical, logistical, and safetystandpoints. Mucosal immune responses are also important forthe prevention of many infectious diseases because they rep-resent the first barrier from the hosts that pathogens mustevade.

Research into the host immune response to pneumococ-cal diseases has focused primarily on the role of innate andadaptative humoral immune responses. However, in the lastfew years, attention has been drawn to cellular immuneresponses against Streptococcus pneumoniae, with interest-ing results. The majority of these studies analyzed cellularaspects of innate immunity and proposed that lymphocytes,neutrophils, and macrophages orchestrate effective immuneresponses without the presence of specific antibodies. In thiscontext, proinflammatory cytokines promote an adequatemilieu for pneumococcal clearance (22, 24, 25, 31, 34, 50,54). A Th1-biased immune response has also been shown tobe engaged in the resolution of pneumococcal infection inhumans (21). Nevertheless, inflammatory cell influx into thelung and mucosal responses must be regulated to avoidexacerbated tissue injury. This is evidenced in recent studiesof 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

* Corresponding author. Mailing address: Centro de Biotecnologia,Instituto Butantan, Av. Vital Brasil 1500, 05503-900 Sao Paulo, Brazil.Phone: 55-11-3726-7222, ext. 2244. Fax: 55-11-3726-1505. E-mail forM. L. S. Oliveira: [email protected]. E-mail for E. N. Miyaji:[email protected].

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

� Published ahead of print on 11 March 2009.

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disease and colonization were shown using pneumococcalwhole-cell vaccines (28, 46) or recombinant proteins as muco-sal vaccines (2, 6, 7, 9, 40). In recent approaches, lactic acidbacteria (3, 11, 18, 37), which are able to activate and modulatethe innate immune system (35, 42), were used for pneumococ-cal antigen presentation, with promising results.

Very few works compared pulmonary and systemic immuneresponses induced by pneumococcal antigens using parenteraland mucosal immunizations (13). The present study aims atinvestigating local and systemic cellular and humoral immuneresponses required for protection against invasive intranasal(i.n.) challenge with S. pneumoniae strain ATCC 6303 usingPspA and PspC antigens administered by both routes, withoutthe use of adjuvants, or presented by Lactobacillus caseithrough the nasal route.

MATERIALS AND METHODS

Bacterial strains and growth conditions. Lactococcus lactis cultures weregrown in M17 medium (Difco); L. casei CECT5275 cultures were grown inMan, Rogosa, and Sharpe medium (Difco); and S. pneumoniae ATCC 6303cultures (serotype 3, PspA clade 5) were grown in Todd-Hewitt broth (Sigma)supplemented with 0.5% yeast extract (THY). Bacteria were grown at 37°Cwithout shaking. Pneumococcal strain ATCC 6303 was always plated in bloodagar and grown overnight at 37°C before inoculation in THY. All bacterialstocks were maintained at �80°C in their respective media containing 20%glycerol.

Recombinant proteins. Expression and purification of the N-terminal frag-ment of PspA from clade 5 (from strain 122/02, serotype 23F) were per-formed as previously described (14). The fragment encoding the PspC N-terminal region was amplified from S. pneumoniae strain 491/00 (InstitutoAdolfo Lutz, Sao Paulo, Brazil) using forward primer 5�-TAGGGATCCCATGCGACAGAGAACGAGA-3� and reverse primer 5�-CTGCAGTTATTGTGGTTGTTCAGC-3�. The gene product was cloned into a pGEMT-Easyvector (Promega), and the sequence was confirmed by DNA sequencing. Thefragment was subcloned into linearized vector pAE (43) and used to trans-form Escherichia coli BL21(DE3) SI competent cells (Invitrogen). Proteinexpression was induced in mid-exponential-phase cultures by the addition of300 mM NaCl. The recombinant proteins bearing N-terminal histidine tagswere purified from the soluble fraction by affinity chromatography usingNi2�-charged resin (HisTrap HP; GE Healthcare). Elution was carried outwith 250 mM imidazole. The purified fractions were analyzed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, dialyzed against 10 mMTris-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-terminalregion of PspA5 and PspC in L. casei. The following primers were used foramplification: 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 vectorpT1NX, and ligation products were used to transform competent L. lactiscells as previously described (37). Plasmids isolated from L. lactis were thenused for the electroporation of L. casei (37). L. lactis and L. casei transfor-mants were selected by plating onto the respective medium containing 1.8%agar and 5 �g/ml of erythromycin. Protein expression was confirmed byWestern blotting of L. casei lysates using specific antibodies.

Immunization of mice. Five- to seven-week-old female C57BL/6 mice from theCentral Animal Facility of Butantan Institute were supplied with food and waterad libitum. Animal experimental protocols were approved by Use Ethics Com-mittee of Instituto Butantan (Sao Paulo, Brazil). Groups of 4 to 10 animals wereanesthetized through the intraperitoneal (i.p.) route with 200 �l of a mixture of0.5% xylazine and 0.2% ketamine and inoculated i.n. with 6 doses (5 �g in 10 �l)on days 0, 3, 14, 17, 28, and 31 or subcutaneously (s.c.) with 3 doses on days 0,14, and 28 (5 �g in 100 �l) of PspC or PspA5 previously treated with TritonX-114 to remove lipopolysaccharide as described previously (1) and without theuse of adjuvants. L. casei cells expressing PspC or PspA5 or carrying the emptyvector 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 �l. 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 immuniza-tion. Vaginal washes were collected from days 15 to 19 after the last immuniza-tion by gentle pipetting of 25 �l of saline twice, and samples were pooled for eachanimal. Antibody levels in serum, vaginal washes, and bronchoalveolar lavagefluid (BALF) (collected as described below) were evaluated by enzyme-linkedimmunosorbent assay (ELISA) in plates coated with PspC or PspA5. The assaywas performed using goat anti-mouse immunoglobulin G [IgG], IgA, IgG1, orIgG2a 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 significantlydifferent. Titers were defined as the last dilution in which absorbances at 492 nmreached 0.1.

Antibody binding and complement deposition assays. Frozen stocks of S.pneumoniae ATCC 6303 were plated onto blood agar overnight and then grownin THY to an OD600 of 0.4 to 0.5 (�108 CFU/ml) and harvested by centrifuga-tion. Bacteria were washed, resuspended in phosphate-buffered saline (PBS),and incubated with 10% of pooled sera during 30 min at 37°C. Samples werewashed once with PBS before incubation with fluorescein isothiocyanate-conju-gated anti-mouse IgG (Sigma) for 30 min on ice. For complement depositionassays, sera were previously heated at 56°C for 30 min and incubated withbacteria at a concentration of 25% at 37°C for 30 min. Samples were washedonce with PBS and incubated with 10% normal mouse serum as the source ofcomplement in Gelatin Veronal buffer (Sigma) at 37°C for 30 min. After wash-ing, samples were incubated with fluorescein isothiocyanate-conjugated anti-mouse C3 IgG (MP Biomedicals) in PBS for 30 min on ice. Samples were fixedwith 2% formaldehyde after two washing steps and stored at 4°C. Flow cytometryanalysis was conducted using a FACSCalibur apparatus (Becton Dickinson), and10,000 gated events were recorded. The median of fluorescent bacteria was usedto compare the groups.

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

Collection of BALF and cytospin. Mice were sacrificed 13 h after i.n. pneu-mococcal challenge by injection of a lethal dose of urethane (15 mg per 10 g ofbody weight) to collect the spleen, lung, and BALF samples. A catheter wasinserted into the trachea of the mice, and lungs were rinsed with 0.5 ml of sterilePBS, followed by an additional rinse with 1 ml of PBS. The fluids from bothrinses were pooled. Cells obtained from the pooled fluid were washed andresuspended in PBS and counted, and 4 � 104 cells were spun onto glass slides(4 min at 1,300 rpm) by the use of a cytocentrifuge (StatSpin Cytofuge). Cytospinslides were stained, and 100 cells were differentially counted to analyze thepercentage of infiltration of each cell type. Fluid samples were stored at �80°Cfor subsequent analyses.

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

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

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RESULTS

Infiltration of cells after i.n. pneumococcal challenge inPspC-immunized mice. Mice were i.n. immunized with thePspC protein without the use of an adjuvant or L. casei cellsexpressing this antigen. Animals immunized with L. casei cellscarrying the empty vector (L. casei) or nonimmunized animalswere used as control groups. An additional group receivedPspC s.c. Animals were bled 15 days after the last immuniza-tion, and antibodies in sera and vaginal washes were analyzedthrough ELISA. Specific anti-PspC IgGs were detected only insera from s.c.-immunized animals (IgG titer of 1:800). For theother groups, no reactivity was observed even at the lowestdilution tested (1:20). In addition, anti-PspC IgA was not ob-served in vaginal washes collected from immunized animals(the minimal dilution tested was 1:4). Twenty-one days afterthe last immunization, all groups were challenged with strainATCC 6303 through the i.n. route, and 13 h after challenge,BALF and tissue samples were collected. The time point of13 h was chosen because at this time, the animals do not yetshow any sign of the disease, and there is no detectable CFUin blood. In addition, no differences in lung CFU counts amongall groups were observed at this point (data not shown). Even13 h after challenge, anti-PspC antibodies were not detected inBALF samples by ELISA. Analysis of cells recovered fromBALF (Fig. 1) showed a significant infiltration of neutrophilsin L. casei PspC-immunized animals compared with the L.casei control group (P 0.0004). Nasal immunization withPspC also induced an augmentation in neutrophil infiltrationcompared with neutrophil infiltration in nonimmunized ani-mals, but this difference was not statistically significant. Nodifference was observed in the percentages of neutrophils re-covered from mice s.c. immunized with PspC compared withnonimmunized animals.

IFN-� and IL-17 secretion by lung and spleen cells in PspC-immunized mice. After collection of BALF, lung and spleenwere excised from mice. Secretion of IFN-� and IL-17 frompooled cells of each group was detected through ELISPOTassay and sandwich ELISA, respectively. As observed in Fig.2A, nasal immunizations with L. casei PspC and PspC wereable to induce an increase in the number of lung cells secretingIFN-� compared with control groups or even with the group

immunized s.c. with PspC. This effect was more pronouncedfor L. casei PspC than for PspC nasal immunization. IFN-�secretion from spleen cells was also analyzed, and PspC im-munization was able to induce an increase in numbers of SFCswhen 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 wasobserved in the group i.n. immunized with PspC (Fig. 2B). Asfor IL-17, all immunized groups displayed elevated levels ofsecretion of this cytokine in lung cell culture supernatants. Themost pronounced secretion was observed in the group immu-nized i.n. with PspC that presented an eightfold increase in theconcentration of this cytokine (Fig. 2C). On the other hand,this immunization induced fourfold-less IL-17 secretion thanL. casei PspC immunization in spleen cells, whereas subcuta-neous immunization with PspC did not produce any increase atall (Fig. 2D).

Serum and mucosal anti-PspA antibody responses. The lev-els of anti-PspA antibodies were measured by ELISA. Asshown in Fig. 3A, i.n. immunization of mice with PspA5 elic-ited significantly higher IgG responses than in nonimmunizedmice (P 0.0001). L. casei PspA5 immunization also led to asignificant increase in levels of anti-PspA antibodies comparedwith those found with L. casei immunization (P 0.002). s.c.immunization with PspA5 induced an elevated level of anti-body production compared with that of nonimmunized animals(P 0.001), but the level was slightly lower than the oneobserved for mice immunized i.n. with PspA5. i.n. immuniza-tion induced intermediate anti-PspA5 IgG1/IgG2a ratios inprotein groups (IgG1/IgG2a ratio of 91) and lower ratios whenthe antigens were delivered by L. casei (IgG1/IgG2a ratio of0.1) (Fig. 3B). The highest ratio was observed in the groupimmunized with PspA5 s.c. (IgG1/IgG2a ratio of 358). Specificanti-PspA secreted IgA (sIgA) was detected only in vaginalwashes obtained from mice i.n. immunized with PspA5 (Fig.3C). These antibodies were not detected in samples from theother groups, even at a 1:4 dilution.

Binding of anti-PspA antibodies and complement deposi-tion onto ATCC 6303. In order to investigate whether anti-PspA antibodies detected by ELISA would be able to bind tothe surface of intact pneumococci and mediate C3 deposition,sera from mice immunized i.n. or s.c. with PspA5 or immu-nized i.n. with L. casei PspA5 were incubated with strainATCC 6303 in an antibody binding assay. Sera from nonim-munized mice or mice immunized with L. casei were used ascontrols. As shown in Fig. 4A, we observed that L. casei PspA5immunization elicited antibodies that showed the highest bind-ing capacity, which was especially surprising compared with s.c.immunization with PspA5. Furthermore, when the ability tomediate C3 deposition onto the pneumococcal surface wasanalyzed (Fig. 4B), sera from animals i.n. inoculated with L.casei PspA5 or PspA5 elicited a more pronounced increase inC3 deposition than did sera from mice s.c. immunized withPspA5. It is important that PspA5 immunization through thes.c. route, without adjuvant, elicited larger amounts of specificPspA5 antibodies than did the L. casei PspA5 group (Fig. 3A).However, these Igs did not display the same efficacy in bindingonto the pneumococcal surface and enhancing complementdeposition.

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

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Enhancement of infiltration of neutrophils and presence ofanti-PspA5 antibodies in BALF after i.n. pneumococcal chal-lenge. BALF cell samples were recovered from animals immu-nized with PspA5 vaccines 13 h after a respiratory challengewith strain ATCC 6303. A significant increase in the level ofinfiltration of neutrophils was observed in mice immunizedwith L. casei PspA5 compared with mice immunized with L.casei (P 0.005) (Fig. 5). BALF recovered from mice immu-nized i.n. with PspA5 also demonstrated an increase in levels ofthese 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 antibodytiter of 1.12 � 0.51) in BALF recovered after pneumococcalchallenge (data not shown).

Cytokine secretion by lung and spleen cells in PspA5-immu-nized mice. Analyzes of the cellular immune response showedthat L. casei PspA5 immunization led to an intense increase inthe 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 PspA5s.c., followed by PspA5 i.n. immunization (Fig. 6B). Con-versely, IL-17 secretion was augmented in lung cells obtainedfrom the groups immunized with PspA5 s.c. and PspA5 i.n.

(Fig. 6C). This last group presented the most pronouncedincrease in levels of IL-17 secretion in the supernatant of lungand spleen cells (Fig. 6D). The level of IL-5, a Th2 cytokine,was also measured, and IL-5 was detected only in the groupimmunized s.c. with PspA5 (Fig. 6E). Additionally, the groupimmunized with PspA5 i.n. displayed the lowest level of secre-tion of the proinflammatory cytokine TNF- by lung cells 13 hafter pneumococcal challenge (Fig. 6F), indicating a controlledinflammatory response.

Evaluation of protection conferred by PspC and PspA vac-cines against invasive i.n. challenge. Mice immunized with thedifferent PspC- and PspA-based vaccines were challenged 21days after the last immunization with strain ATCC 6303through the i.n. route and were sacrificed 13 h later for deter-minations of CFU in lungs or were monitored during 10 daysfor observation of survival. No significant changes in CFUcounts in the lungs were observed among all the groups 13 hafter challenge (data not shown). By analyzing PspC-basedvaccines, we observed that despite the cellular immune re-sponses elicited, no significant survival was observed in miceimmunized with any formulation or by any administrationroute (Table 1). Sequence comparison of the N-terminal re-gion of the PspC molecule used in our vaccines and ATCC6303 PspC showed 47% amino acid conservation (identical

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 withstrain ATCC 6303. Spleen and lung cells were isolated from immunized mice 13 h after i.n. challenge and incubated with PspC in ELISPOT platespreviously coated with anti-IFN-�. SFCs of IFN-� were detected through ELISPOT, and the average number of spots in duplicate wells wascalculated by subtracting nonstimulated wells and considered as the number of SFCs/106 cultured cells (A and B). Cells were also incubated withPspC, and IL-17 secretion in the supernatants was detected through sandwich ELISA (C and D). Results are representative of two independentexperiments. L.c., L. casei; Non, nonimmunized group.


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plus conserved residues). Vaccines composed of PspC mole-cules with higher degrees of conservation remain to be testedin this model. On the other hand, as shown in Table 2, i.n.PspA5 and L. casei PspA5 immunizations were able to confersignificant protection against this invasive pneumococcal chal-lenge compared with nonimmunized mice (60% for mice im-munized with PspA5 i.n. and 40% for mice immunized with L.casei PspA5 i.n.) (P 0.002 and P 0.02, respectively, byFisher’s exact test). Through this type of analysis, s.c. admin-istration of PspA5 was not able to confer protection againstthis challenge model. Kaplan-Meier analysis confirmed signif-icant protection in mean survival times for animals i.n. immu-

nized with PspA5 (P 0.0006) and L. casei PspA5 (P 0.005)compared with nonimmunized mice. An increase in mean sur-vival time was also observed in animals s.c. immunized withPspA5 (P 0.04) (Fig. 7). The L. casei control group did notelicit significant protection in this pneumococcal challengemodel neither in total survival rate (P 0.21) nor in meansurvival time analysis (P 0.15).

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

DISCUSSION

The precise mechanisms of the resolution of S. pneumoniaelung infection conferred by effective mucosal vaccines have notyet been elucidated. In the present study, we used two of themost promising pneumococcal vaccine candidates, PspC andPspA, through different routes of immunization (mucosal andparenteral) and also used the lactic acid bacterium L. casei asa delivery system of these antigens in order to evaluate theinduction of pulmonary and systemic immune responses andtheir protective potential against an invasive challenge with S.pneumoniae in mice.

The investigation of PspC-based vaccines revealed that thegroup vaccinated with L. casei expressing this antigen was theonly group that presented a significant enhancement in theinfiltration of neutrophils in BALF compared with the controlgroup inoculated with L. casei. As for the humoral response,antibodies against PspC were observed only in sera from mices.c. immunized with the recombinant protein. The better effi-cacy of the s.c. route over the mucosal route of immunizationfor the induction of anti-PspC IgG in sera may be a conse-quence of differences in antigen capture and presentation aswell as protein stability in each environment, favoring s.c. im-munization. None of the formulations were able to elicit mu-cosal antibodies under the conditions tested. Immunizationwith recombinant PspC through different routes has beenshown to induce specific antibodies and to confer protectionagainst different pneumococcal challenge models (7, 12, 36).The absence of anti-PspC antibodies in mice immunized withour PspC protein-based vaccines maybe explained by the factthat we have removed residual lipopolysaccharides resultantfrom E. coli purification and tested them in the absence ofadjuvants. It is also important that the experiments describedin the present work were performed using C57BL/6 mice,whereas previous works used BALB/c mice. It is known thatthe susceptibilities of these two mouse strains to pneumococcalinfection are different and may also have contributed to thedifferences observed (19).

The cellular immune response was characterized by the se-cretion of IFN-� and IL-17 by lung and/or spleen cells. Despitethe detection of antigen-specific cellular immune responses,protection was not observed in PspC-immunized mice. How-ever, further studies to analyze the protective capacity of vac-cines composed of PspC molecules displaying a higher degreeof conservation with the PspC-expressed ATCC 6303 must beconsidered.

FIG. 3. Evaluation of anti-PspA antibodies in mice immunized withPspA5 vaccines. Two weeks after the last immunization, anti-PspA IgG(A), IgG1, and IgG2a (B) in sera and IgA in vaginal washes (C) weredetected through ELISA. Log10 values of antibody titers are shown,and IgG1/IgG2a titer ratios are indicated above the bars (B). Resultsare representative of two experiments. Asterisks represent significantdifferences 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.


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In our hands, PspA5 was shown to be more immunogenicthan PspC. Antibodies elicited by all PspA5 formulations wereable to bind to strain ATCC 6303. Even with lower levels ofIgG induced by L. casei PspA5, serum obtained from thisgroup showed superior binding compared with those of serafrom other groups. These results may be explained by the factthat the antigen expressed and delivered by L. casei may havea more appropriate folding, resulting in antibodies that recog-nize epitopes present in the PspA molecule attached to thepneumococcal surface.

Nasal PspA5 vaccines displayed higher levels of IgG2a thandid the s.c. vaccine, which was reflected in more balancedIgG1/IgG2a ratios. Nevertheless, the L. casei PspA5 group wasthe only group that displayed larger amounts of IgG2a thanIgG1. These isotyping results are in accordance with previouswork with lactic acid bacteria showing that L. lactis adminis-tration through the i.n. route induced a Th1-biased immuneresponse with higher levels of IgG2a production, while itsadministration though the i.p. route led to the production ofelevated amounts of IgG1 (45). It has also been reported thatlactobacilli may facilitate the polarization of the naive immunesystem by skewing it away from Th2 responses and toward Th1immune responses (35).

When evaluating complement deposition, we observedthat the groups immunized i.n. with L. casei PspA5 and PspA5presented the greatest ability in mediating complement depo-sition onto the pneumococcal surface. Interestingly, comple-ment deposition observed with sera from L. casei PspA5-im-munized mice was similar to that observed for sera from miceimmunized i.n. with PspA5 despite the smaller amounts ofantibody elicited. These results can be explained by the factthat the lactobacillus vaccine elicited predominantly IgG2a, incontrast with the other groups. In a previous work by ourgroup, we proposed that since anti-PspA IgG2a would be theisotype with the greatest capacity to mediate complement dep-osition onto the pneumococcal surface, large amounts of IgG1(in this case elicited by s.c. immunizations) could compromisecomplement deposition by impairing the binding of IgG2aantibodies (17). Arulanandam and colleagues have also shownthat Th1 immune responses, as the one elicited by PspA using

FIG. 4. Binding of anti-PspA5 antibodies and complement deposition onto the pneumococcal surface. Sera from mice i.n. immunized withPspA5 (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 thepneumococcal surface (A) and to mediate C3 deposition (B). S. pneumoniae ATCC 6303 was incubated with 10% (A) or 25% (B) serum from eachgroup 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, fluoresceinisothiocyanate.

FIG. 5. BALF cell counts. Slides were prepared with 4 � 104 cellsfrom BALF. Percentages of infiltrated cells are expressed as means ofdata from four to five mice per group. An � represents a significantdifference in neutrophil infiltration between mice immunized i.n. withPspA5 and nonimmunized mice (Non). �� represents a significantdifference in neutrophil infiltration from mice immunized with L. caseiPspA5 and L. casei (L.c.) (�, P 0.001; ��, P 0.005 [Mann-WhitneyU test]).


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IL-12 as an adjuvant through the i.n. route, led to the produc-tion of large amounts of IgG2a and IgA and correlated thisincrease with protection against lung infection. Blocking theseIgs with specific proteins significantly inhibited pneumococcaluptake by phagocytic cells (2).

i.n. immunization with PspA5 also induced specific sIgAantibodies detected in vaginal washes 2 weeks after the lastimmunization and in BALF samples 13 h after challenge. Thisfinding correlated with better protection in our model. Therole of sIgA against colonization by S. pneumoniae in nasalmucosa is controversial. While some studies demonstrated thatprotection can occur in the absence of B cells (30, 31), a crucial

role for sIgA was demonstrated in a model using pIgR�/� andIgA�/� mice (49). Still, it is well known that sIgA exerts pro-tection by noninflammatory mechanisms such as the inhibitionof bacterial adhesion or opsonization not mediated by comple-ment (39). Such a response may therefore contribute to thesurvival of mice.

The highly encapsulated strain ATCC 6303 (serotype 3) isextremely virulent to mice in the i.n. challenge used in thisstudy. In vitro studies have shown that neither human nor ratpolymorphonuclear cells are able to phagocytose this strain (8,16). Even with this high virulence, in this study, we showed a

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 PspA5in ELISPOT plates previously coated with anti-IFN-�. SFCs of IFN-� were detected through ELISPOT assay, and the average number of spotsin duplicate wells was calculated by subtracting nonstimulated wells and considered as the number of SFCs/106 cultured cells (A and B). Cells werealso 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.

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

Group No. of alive mice/total no. of mice

%Survival P

Nonimmunized 2/10 20PspC 1/8 12.5 0.49L. casei 1/8 12.5 0.49L. casei PspC 2/10 20 0.49PspC s.c. 0/11 0 0.2

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

Group No. of alive mice/total no. of mice

%Survival Pa

Nonimmunized 0/13 0PspA5 6/10 60 0.002L. casei 3/13 23 0.21L. casei PspA5 4/10 40 0.02PspA5 s.c. 3/12 25 0.09

a Fisher exact test.


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significant protection of mice i.n. immunized with PspA5(60%) or with L. casei PspA5 (40%) compared with nonim-munized animals. It is important that the pspA5 fragment usedin this study was amplified from a Brazilian pneumococcalisolate (strain 122/02, serotype 23F) (14) and not from thesame strain used for the challenge. Using the s.c. route, pro-tection was observed only by mean survival time analysis, show-ing that i.n. immunization was important to elicit an adequateimmune response required in this challenge model.

Elevated levels of IFN-� and low levels of IL-17 secretion bylung cells were observed after immunization with L. caseiPspA5. The opposite (high levels of IL-17 and low levels ofIFN-�) was observed for i.n. immunization with PspA5. Theprotective role of IL-17 in a mouse model of pneumococcalcolonization was very well characterized by the works of Malleyand collaborators. In those studies, the authors elegantlyshowed the role of IL-17-expressing CD4� T cells in the pro-tection conferred by a killed whole-cell vaccine. However, allthe correlations determined to date are related to protectionagainst the pneumococcal nasopharyngeal colonization model(27, 29, 52). IL-17 acts on the recruitment of neutrophils tosites of infection and is also involved in pulmonary host de-fenses against various pathogens (4, 5, 41). Nevertheless, wehave no knowledge of data on IL-17 expression by lung cells inmouse models of pneumococcal infection. In our model, in-creases in levels of IL-17 secretion and neutrophil infiltrationafter challenge were observed for i.n. immunization withPspA5, correlating with the highest protection level. In L. caseiPspA5-immunized mice, neutrophil infiltration was also ob-served, but no IL-17 secretion was observed 13 h after chal-lenge, suggesting different mechanisms for protection elicitedby the two vaccines.

It was recently reported that i.n. IL-12 administration innaive mice led to IFN-� production by NK cells and TNF-production by macrophages in the lung, increasing neutrophil

recruitment (50). Nonrecombinant lactobacilli are known toinduce high levels of IL-12 release, inhibiting Th2 cytokineresponses (IL-4 and IL-5) (35). Although a Th1-biased im-mune response seems to be a key component of the hostdefense against invading pathogens such as pneumococcus,this polarized immune response has also been implicated ininflammatory disorders (32, 33) and may cause lung injury(48). Secretion of IFN-� by lung cells and protection againstpneumonia were described previously (50, 54). On the otherhand, in some cases, secretion of this cytokine seems to bedetrimental (44, 47). In a mouse model of pneumonia, it hasbeen shown that elevated levels IFN-� secreted by NK cellspresent in lungs of scid mice are unfavorable for recoveringfrom infection (23). Accordingly, the high levels of IFN-� re-leased by lung cells observed in the L. casei PspA5-immunizedgroup may be prejudicial, resulting in only 40% protection.Other aspects of such polarized immune response are benefi-cial, such as the large amounts of IgG2a, leading to enhance-ment in complement deposition.

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

Although no histological signs of inflammatory injuries wereobserved 13 h after challenge, a possible deleterious effect, inadvanced stages, of the intense release of TNF- observed incontrol groups as well as in groups i.n. immunized with L. caseiPspA5 or s.c. immunized with PspA5 cannot be discarded. Thegroup of mice i.n. immunized with PspA5 was the only groupwith low levels of TNF- release by lung cells 13 h afterchallenge, and this was exactly the group that presented thebest protection.

Protection using lactic acid bacteria as an antigen deliverysystem was previously reported with L. lactis or L. casei cellsexpressing PspA against respiratory or i.p. challenge with dif-ferent pneumococcal strains (11, 18). However, those previousworks did not clarify the mechanisms involved in protectionagainst S. pneumoniae. The present work is the first to aim atcharacterizing both humoral and cellular immune responsesinduced locally (lung) and systemically (spleen) by the admin-istration of a pneumococcal antigen through a lactic acid bac-terium delivery system. Furthermore, quantitative and qualita-tive features of mucosal and s.c. immunizations have beenidentified. Differences between the two routes of administra-tion using two promising protein vaccine candidates were eval-uated. The bias toward the Th1 immune response obtained byi.n. immunization with PspA5 is of particular interest sinceIFN-� as well as IgG2a are critical in defense mechanismsagainst pneumococci. A concern about the administration ofantigens through the i.n. route is the need for a safe and goodadjuvant (38). Different tests showed the difficulty in maintain-ing adjuvanticity while reducing toxic effects of an adjuvantmolecule (15). In this work, we showed that PspA5 is highlyimmunogenic and is able to induce protective humoral andcellular immune responses through i.n. administration without

FIG. 7. Mean survival time of mice after i.n. challenge. Immunizedmice were challenged with 105 CFU of pneumococcal strain ATCC6303 through the i.n. route, and survival was monitored for 10 days.Results were evaluated by Kaplan-Meier survival curve analysis. A Pvalue of �0.05 was considered to be significantly different (�). L.c., L.casei.


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the use of adjuvant. We thus propose that the protectionagainst this invasive challenge is likely due to both complementdeposition induced by specific anti-PspA5 IgG2a and elevatedpulmonary immunity with secretion of proinflammatory cyto-kines. Nevertheless, control of TNF- cytokine release is im-portant to increase protection.

ACKNOWLEDGMENTS

We are deeply grateful to Ana Paula Christ for help with the im-plementation of BALF collection protocols and Vania Gomes de Mat-taria for the animal facilities coordination.

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

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characterization of protective mucosal and systemic ...(20). immunization via mucosal surfaces would...

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