ABSTRACT
Pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC) are important candidates for an alternative vaccine against pneumococcal infections. Since these antigens show variability, the use of variants that do not afford broad protection may lead to the selection of vaccine escape bacteria. Epitopes capable of inducing antibodies with broad cross-reactivities should thus be the preferred antigens. In this work, experiments using peptide arrays show that most linear epitopes recognized by antibodies induced in mice against different PspAs were located at the initial 44 amino acids of the mature protein and that antibodies against these linear epitopes did not confer protection against a lethal challenge. Conversely, linear epitopes recognized by antibodies to PspC included the consensus sequences involved in the interaction with human factor H and secretory immunoglobulin A (sIgA). Since linear epitopes of PspA were not protective, larger overlapping fragments containing 100 amino acids of PspA of strain Rx1 were constructed (fragments 1 to 7, numbered from the N terminus) to permit the mapping of antibodies with conformational epitopes not represented in the peptide arrays. Antibodies from mice immunized with fragments 1, 2, 4, and 5 were capable of binding onto the surface of pneumococci and mediating protection against a lethal challenge. The fact that immunization of mice with 100-amino-acid fragments located at the more conserved N-terminal region of PspA (fragments 1 and 2) induced protection against a pneumococcal challenge indicates that the induction of antibodies against conformational epitopes present at this region may be important in strategies for inducing broad protection against pneumococci.
INTRODUCTION
Streptococcus pneumoniae is a major cause of morbidity and mortality due to pneumonia, meningitis, and bacteremia. It has been estimated that 14.5 million episodes of serious illnesses caused by pneumococci occurred worldwide in 2000, leading to the death of 826,000 children <5 years of age. Infections caused by pneumococci were therefore responsible for 11% of all deaths in this age group (1). The capsular polysaccharide (PS) is the most well-known virulence factor of S. pneumoniae, being involved in the evasion of complement deposition and phagocytosis during invasion of the human host. The currently available vaccines are based on the induction of anti-PS antibodies, and the protein-PS conjugate vaccines are very efficient at preventing invasive disease (especially bacteremia and sepsis) caused by capsular serotypes included in their formulations. More than 90 capsular serotypes with limited cross-reactivities have been described, and the widespread use of conjugated vaccines has led to an increased rate of disease caused by nonvaccine serotypes, a phenomenon known as serotype replacement (2–4). Conjugated vaccines are expensive, which limits their implementation in national immunization programs in most countries.
A vaccine strategy for avoiding capsular serotype replacement can utilize protein antigens, such as pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC), which have been shown to elicit protective antibodies against pneumococci (5–8). PspA interferes with the deposition of complement onto the surface of pneumococci (9, 10), has an antiphagocytic effect that does not depend on complement (11), and inhibits death mediated by apolactoferrin at mucosal sites (12). Mature PspAs are composed of three major domains, an α-helical amino-terminal domain, a proline-rich domain, and a choline-binding domain that anchors the protein to the cell wall (13–15). PspAs show variation between strains in the N-terminal surface-exposed region and are classified into 1 of 6 clades according to amino acid divergence in the clade-defining region (CDR), which comprises the C-terminal third of the N-terminal domain (13). The clades are grouped in 3 families; family 1 includes clades 1 and 2, family 2 includes clades 3, 4, and 5, and family 3 is rarely isolated and includes only clade 6 (13). Even within clades, two PspAs are seldom identical, although PspAs of the same clade or family are generally very cross-reactive (6, 13, 16, 17). Thus, a logical PspA-containing vaccine should include representatives from each of the two major families (6, 16, 17).
PspC, also called choline-binding protein A (CbpA) (18), acts as an adhesion molecule through interaction with the secretory component of human IgA (19) and to the laminin receptor (20), and it is capable of interacting with complement through binding to C3 (21) and factor H (FH) (22–25). The interaction of PspC with C3 and FH seems to further increase adhesion to epithelial cells (26, 27). PspC is composed of an N-terminal α-helical domain exposed at the surface of the bacteria, followed by a proline-rich region and a cell surface anchoring motif (28). PspC molecules show variability between strains and are classified in 11 groups (29).
Recent studies showed that, in addition to the region containing the genes for capsule synthesis, the pneumococcal genetic loci of surface antigens that demonstrated the greatest selective pressure by the highest recombination rates were pspA, pspC, and psrP (pneumococcal serine-rich repeat protein) (30). It was therefore proposed that these antigens are really important and are involved in evasion of the immune system, but their use as a vaccine could lead to a replacement phenomenon similar to that observed with PS-conjugated vaccines. Thus, the choice of PspA and PspC molecules capable of inducing antibodies with broad cross-reactivities is essential. We previously showed that PspA from clade 4 (PspA4), PspA from clade 5 (PspA5), and PspC from group 3 (PspC3) induced antibodies that recognized the majority of the pneumococcal clinical isolates tested (31–33). Alternatively, cross-reactive immunogenic epitopes present in PspA and PspC can be selected to compose a multiepitope protein vaccine.
In this work, we used a screening method with peptide arrays containing 15-mer peptides covering the entire sequence of different PspAs and PspC3 to analyze sera immune to these antigens. We previously used a similar method to compare sera from mice immunized with one PspA variant as a recombinant protein and as a DNA vaccine (34). This technique has also been used successfully to screen epitopes of antigens from other pathogens (35, 36). The present study tested sera from mice immunized with several variants of PspA and PspC3 to identify the most immunogenic epitopes. We also evaluated the protective capacity of antibodies against immunogenic linear epitopes. We localized the PspA regions capable of inducing protective immunity using 100-amino-acid fragments of PspA to permit the detection of conformational epitopes. Our findings have important implications for vaccine development, providing insight into the protective capacity of antibodies against both linear and conformational epitopes of PspA.
MATERIALS AND METHODS
Expression of recombinant proteins.The plasmids for the expression of PspAs (from the mature N terminus to the proline-rich region) from clade 1 (PspA1; strain 435/96), clade 2 (PspA2; strain 371/00), clade 3 (PspA3; strain 259/98), clade 4 (PspA4; strain 255/00), clade 5 (PspA5; strain 122/02) (31, 32), and PspC group 3 (PspC3; strain 491/00) (37) were previously constructed in our laboratory. pspARx1 and its fragments (fragments 1 to 7) were amplified by PCR from strain Rx1 and cloned into the pAE vector (38) for expression in Escherichia coli with an N-terminal histidine tag, using the primers listed in Table S1 in the supplemental material, generating pAE-pspARx1 (1,011 bp), pAE-frag1 (300 bp), pAE-frag2 (300 bp), pAE-frag3 (300 bp), pAE-frag4 (300 bp), pAE-frag5 (300 bp), pAE-frag6 (300 bp), and pAE-frag7 (243 bp). The 100-amino-acid fragments cover the mature N-terminal region to the proline-rich region of PspARx1 with a 50-amino-acid overlap. The synthesis of the plasmid containing repeats of part of the N-terminal region of pspA4 (pspAN-terminal-repeat; 402 bp) (see Fig. S1 in the supplemental material) (pUCAmp-pspAN-terminal-repeat) was carried out by Blue Heron Biotechnology (Bothell, WA, USA). The gene is composed of three repetitions of codons encoding amino acids 6 to 44 of the N-terminal region of mature PspA4. Six glycines were used between each repetition (linker). The restriction sites of the XhoI and EcoRI enzymes were included at the beginning and at the end of the sequence, respectively, for cloning in the pAE vector. The plasmid pUCAmp-pspAN-terminal-repeat was digested with XhoI and EcoRI, and the gene was subcloned into the pAE vector, generating pAE-pspAN-terminal-repeat. All cloning procedures were performed in E. coli DH5-α grown in Luria-Bertani medium supplemented with ampicillin (100 μg/ml). All proteins were purified through Ni2+ affinity chromatography from the soluble extract from E. coli BL21 SI (Invitrogen).
Pneumococcal strains.S. pneumoniae strains D39 (serotype 2, PspA clade 2), A66.1 (serotype 3, PspA clade 2), 3JYP2670 (serotype 3, PspA clade 4), and ATCC 6303 (serotype 3, PspA clade 5) (13, 39–41) were grown in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY) and maintained as frozen stocks (−80°C) in THY containing 20% glycerol.
Immunization of mice.Our animal experimental protocols were approved by the Instituto Butantan Ethical Committee for Animal Research (São Paulo, Brazil; approvals CEUAIB 457/08 and 1012/13). Five- to 7-week-old female specific-pathogen-free BALB/c mice were obtained from the University of São Paulo Medical School (FM-USP, São Paulo, Brazil). Six mice were given three subcutaneous (s.c.) doses of 5 μg of proteins using aluminum hydroxide (Alum; 50 μg of Al3+) as adjuvant at 14-day intervals. Animals injected with only Alum were used as controls.
Measurement of antibodies by ELISA.Two weeks after the last immunization, the mice were individually bled from the retroorbital plexus. Enzyme-linked immunosorbent assays (ELISA) were carried out as described previously (34).
Antibody binding to live pneumococci.Antibody-binding assays were performed as previously described (42). S. pneumoniae strains were grown in THY to a concentration of 108 CFU/ml (optical density at 600 nm [OD600], 0.4 to 0.5) and harvested by centrifugation at 2,000 × g for 2 min. The pellets were washed with phosphate-buffered saline (PBS), resuspended in the same buffer, and incubated with 1% of the pooled sera for 30 min at 37°C. Samples were then washed with PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (1:1,000) (Sigma) on ice for 30 min. Bacteria were then washed twice with PBS, resuspended in 2% formaldehyde in PBS, and analyzed using FACSCanto (BD Biosciences). Ten thousand gated events were acquired and analyzed in fluorescence intensity histograms using FlowJo 7.6.1. software.
Epitope mapping using peptide arrays.Peptide arrays (CelluSpots, Intavis, Germany) containing 15-amino-acid peptides with an 11-amino-acid overlap covering the N-terminal region of the mature protein to the proline-rich region of PspAs from clades 1, 3, 4, and 5 (PspA1 of strain 435/96 [GenBank accession no. AY082387], PspA3 of strain 259/98 [AY082389], PspA4 of strain 255/00 [EF649969], and PspA5 of strain 122/02 [EF649970]), PspA2 of strain Rx1 (M74122.1), and PspC3 of strain 491/00 (EF424119.1) were purchased from Intavis AG. Two sets of peptide arrays were synthesized, the first containing sequences of PspAs of clades 1, 3, 4, and 5 (see Table S2 in the supplemental material) and the second containing sequences of PspA2 expressed by strain Rx1 and PspC3 expressed by strain 491/00 (see Table S3 in the supplemental material).
For the analysis of antibodies binding to the PspA and PspC3 epitopes, the peptide arrays were first blocked overnight at room temperature with Tris-buffered saline–0.1% Tween 20 (TBS-T)–10% skimmed milk powder. They were then incubated for 5 h at room temperature with pooled sera from mice immunized with Alum, PspA1 plus Alum, PspA2 plus Alum, PspA3 plus Alum, PspA4 plus Alum, PspA5 plus Alum, PspC3 plus Alum, or PspAN-terminal-repeat plus Alum (10 μg/ml of anti-protein IgG), followed by three washes with TBS-T and an incubation with anti-mouse IgG conjugated to alkaline phosphatase (1:5,000) (Sigma-Aldrich). For the Alum control group, incubation was performed with a volume of serum equivalent to the largest volume of anti-PspA serum used in the assays. Detection was performed using 60 μl of 0.12 M 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 50 μl of 0.16 M 5-bromo-4-chloro-3-indolyl phosphate (BCIP), and 40 μl of 1 M MgCl2 in 10 ml of citrate-buffered saline (NaCl 137 mM, KCl 3 mM, citric acid 10 mM) (pH 7.0).
Intranasal challenge.S. pneumoniae A66.1 or ATCC 6303 was grown in THY medium until the OD600 was 0.4 and then aliquoted and kept frozen at −80°C. After this, 3 × 105 (ATCC 6303) or 106 (A66.1) CFU were inoculated with 50 μl into one nostril of each mouse previously anesthetized through the intraperitoneal (i.p.) route with 200 μl of a 0.2% xylazine and 1.0% ketamine mixture 14 days after the last immunization. Survival was monitored for 10 days.
Statistical analysis.The Fisher exact test was used to compare survival levels between groups (GraphPad Prism 5).
RESULTS
Evaluation of linear epitopes recognized by serum antibodies elicited by immunization with the different PspAs.Mice were immunized with PspA1, PspA2, PspA3, PspA4, and PspA5 (representing the α-helical region to the proline-rich domain of PspAs of clades 1 to 5). The concentrations of antibodies induced against these recombinant proteins were determined by ELISA. The sera were then used to probe arrays containing peptides covering the entire sequences of PspA1, PspA3, PspA4, and PspA5. To detect possible nonspecific interactions between the secondary antibody conjugated to alkaline phosphatase and the peptides, a slide was initially incubated with this commercial antibody without any serum from immunized mice. We did not observe any nonspecific reactions (not shown). We performed assays with the peptide arrays using pooled sera from mice injected with Alum alone or immunized with PspA1 plus Alum, PspA2 plus Alum, PspA3 plus Alum, PspA4 plus Alum, or PspA5 plus Alum. Figure 1 shows all of the peptides that were recognized by the tested sera. In general, the sera recognized more peptides in homologous sequences than in heterologous sequences. All the sera recognized epitopes present at a highly conserved first ∼70 amino acids of the N-terminal region of PspA. The anti-PspA2 serum recognized the fewest peptides, and the anti-PspA3 serum recognized the most peptides. Our group previously showed that anti-PspA4 and anti-PspA5 sera were broadly cross-reactive, recognizing pneumococci expressing PspAs from clades 1 to 5 by Western blot analysis (31). We had hoped to identify the epitopes responsible for this broad recognition through analysis of the peptide arrays, but we were not able to determine the basis for this reactivity, owing to the large numbers of peptides detected and the similarity of the patterns observed with the antisera to the 5 different PspAs. Sera raised against PspA4 and PspA5 showed similar recognition patterns, with positive reactivity to peptides located at the N-terminal region (especially within the first 70 amino acids) of homologous (PspA4 or PspA5) and heterologous (PspA1, PspA2, PspA3, and PspA4 or PspA5) PspAs and recognition of only a few peptides in other regions of the homologous molecule. Anti-PspA4 serum was also capable of recognizing a peptide of the nonproline block (NonPro) within the proline-rich region. Sequences of all the peptides recognized by each serum sample are listed in Tables S4 to S8 in the supplemental material.
Schematic illustration of the localization of peptides recognized by all sera in the immunodetection assays with peptide arrays. The mature sequence starts with the fourth amino acid shown (either EEA or DEA, indicated by an arrow). Peptides recognized by anti-PspA1, anti-PspA2, anti-PspA3, anti-PspA4, and anti-PspA5 are shown aligned with the PspA sequences. The localization of the more conserved N-terminal region, the clade-defining region (CDR) of families 1 and 2, and the proline-rich region are indicated.
PspAN-terminal-repeat.Since we did not identify the epitopes responsible for the broad cross-reactivities of anti-PspA4 and anti-PspA5 sera, we decided to construct a gene encoding the N-terminal portion of PspA4, toward which the antibody responses to PspA4 and PspA5 were skewed (pspAN-terminal-repeat). Our goal was to see if this molecular construct would be especially able to induce cross-reactive antibodies and to elicit cross-protection. The synthetic gene encodes three repetitions of the highly conserved amino acids 6 to 44 of the N-terminal region of PspA4 (ASQSKAEKDYDAAMKKSEAAKKAYEEAKKKAEDAQKKYD) (see Fig. S1 in the supplemental material). Six-glycine linkers connected the first and second and the second and third repeats of the PspA sequence, aiming to promote flexibility between the three repetitions.
This 15-kDa recombinant PspAN-terminal-repeat protein was used to immunize mice as recombinant protein plus Alum. The concentrations of antibodies against this recombinant protein were determined by ELISA, and the pooled sera were used to probe the peptide array containing the different PspAs. The anti-PspAN-terminal-repeat serum was capable of recognizing peptides located at amino acids 6 to 44 of all mature PspA sequences, as expected (Table 1). Of the 9 peptides recognized by the antiserum, 8 contained sequences from the immunogen; 4 had the common motif QSKAEKDYD, 3 contained the motif KKAEDAQKKYD, and 1 contained EDQKK. One of the sequences (EIDEEINKAKQILNE) bore no resemblance to the immunizing construct.
Peptides recognized in the first set of arrays by sera from mice immunized with PspAN-terminal-repeat plus Alum
Next, we determined whether antibodies generated against our PspAN-terminal-repeat construct were able to recognize the PspA 1 to 5 constructs containing the mature N-terminal domain to the proline-rich region by ELISA. These antibodies were capable of recognizing each of the 5 large PspA constructs and the immunogen construct PspAN-terminal-repeat (see Fig. S2 in the supplemental material). To determine if this result would be the same for bacteria expressing PspA, the capacities of the antibodies to bind to the surface of intact bacteria were analyzed by flow cytometry. Two different strains were used, ATCC 6303, expressing PspA5, and 3JYP2670, expressing PspA4. The results shown in Fig. 2 indicate that anti-PspAN-terminal-repeat antibodies bound weakly to ATCC 6303 (Fig. 2A) and did not bind at all to 3JYP2670 (Fig. 2B), whereas antibodies elicited to PspA5 and PspA4 readily bound to the bacterial surface. We also tested the binding of antibodies against PspAN-terminal-repeat to a strain with a thinner capsule (255/00, serotype 14, PspA4) and to a nonencapsulated strain (Rx1), but no binding was observed (not shown).
Binding of anti-PspAN-terminal-repeat IgG to intact pneumococci. Pooled sera from mice immunized with PspAN-terminal-repeat were tested for the capacity to bind to the surface of pneumococci. Binding of anti-PspAN-terminal-repeat to strain ATCC 6303 (PspA5) (A) or 3JYP2670 (PspA4) (B) was analyzed (dotted lines). Sera from animals immunized with PspA5 or PspA4 were used as positive controls (continuous lines). Sera from animals injected with Alum alone were used as the negative control (gray areas). The median bacterial fluorescence is shown for each sample. FITC-A, fluorescein isothiocyanate A.
We then evaluated the protective capacity of PspAN-terminal-repeat in immunized mice. The animals were challenged with the pneumococcal strain ATCC 6303, and survival was then monitored for 10 days. None of the immunized mice was able to survive the challenge. Thus, it seems that even though linear epitopes present in the N-terminal region of PspA are recognized by the antibodies in the peptide arrays and in ELISA of the larger PspA molecules, these antibodies do not bind to PspA expressed on the surface of the bacteria, and they are not able to confer protection against a lethal pneumococcal challenge.
PspARx1 fragments of 100 amino acids.Since linear epitopes did not explain the cross-reactivity between the PspAs that we had observed previously and that were not capable of eliciting protection in mice against a lethal pneumococcal challenge, we decided to construct fragments containing 100 amino acids with a 50-amino-acid overlap (fragments 1 to 7), covering the N-terminal region of the mature protein to the proline-rich region of PspA of strain Rx1 (Fig. 3A). Our goal was to immunize with molecules bearing conformational epitopes that were not represented in the peptide arrays. The PspARx1 was chosen because it was the first PspA molecule described and to have its protective domains mapped (43, 44). Mice injected with Alum plus PspARx1 or fragment 1, 2, 3, 4, 5, 6, or 7 developed serum antibodies detected by ELISA (not shown). The sera from mice immunized with the PspA fragments or Alum alone were evaluated by flow cytometry for the capacities of the antibodies to bind to the surface of intact bacteria (Fig. 3B). For this assay, we used the D39 strain, which is the encapsulated parental strain of Rx1 and expresses an identical PspA molecule (45). Antibodies generated against fragments 3, 6, and 7 were not able to bind onto the surface of D39. Antibodies generated against fragments 1 and 2 showed moderate binding onto D39, and antibodies generated against fragments 4 and 5 and against PspARx1 showed strong binding to D39. This assay was performed using 1% serum, but similar results were obtained when equivalent amounts of IgG were used (not shown). Binding of the sera was also tested with pneumococcal strain A66.1 (PspA2), which showed a similar pattern, with greater binding capabilities for the antibodies to fragments 4 and 5, albeit at lower median values, for all sera tested than to D39 (not shown). Immunized mice were then challenged with strain A66.1, and survival was monitored for 10 days. In complete accordance with the data obtained by flow cytometry, the animals immunized with fragments 1, 2, 4, and 5 showed statistically significant higher survival rates than those immunized with Alum, with 100% survival rates for those immunized with fragments 4 and 5 (Table 2). This result is also in agreement with the 100% and 98% identities between PspA from strains Rx1 and A66.1 in fragments 4 and 5, respectively (see Fig. S3 in the supplemental material for the alignments of PspA2 from Rx1 and PspA2 from A66.1). These fragments comprise the clade-defining region and show >90% identity within the same clade and >45% divergence between families (13). Fragments that did not enhance the final survival rate did not augment survival time either (not shown). In order to analyze cross-protection, we also evaluated the survival of mice immunized with the PspA fragments from Rx1 (PspA2) and challenged with ATCC 6303 (PspA5), but no protection was observed.
PspARx1 fragments. (A) Schematic diagram of PspARx1 fragments. On the top is the whole native PspA molecule containing the N-terminal α-helical domain (including regions A and B), the proline-rich region (region C), and the choline-binding domain. Each fragment has 100 amino acids (aas) with a 50-amino-acid overlap (except fragment 7, which has 81 amino acids). The recombinant PspARx1 is also indicated. The initial and final amino acids for each fragment are shown. (B) Binding of anti-PspARx1 and anti-PspARx1 fragment IgG to intact pneumococci. Sera from mice immunized with PspARx1 (α-PspARx1) or its fragments, fragment 1 (α-F1), fragment 2 (α-F2), fragment 3 (α-F3), fragment 4 (α-F4), fragment 5 (α-F5), fragment 6 (α-F6), and fragment 7 (α-F7), were tested for their capacity to bind to the surface of pneumococci. Binding of IgG to strain D39 was analyzed. Sera from animals injected with Alum alone were used as the control (heavy line with light-gray area). The median bacterial fluorescence is shown for each sample.
Survival after lethal intranasal challenge with pneumococcal strain A66.1a
Evaluation of linear epitopes recognized by serum antibodies elicited by immunization with PspC3.PspC3 was also used to immunize BALB/c mice as recombinant protein plus Alum, and the serum was used to probe the array containing peptides covering the N-terminal region to the proline-rich region of mature PspC3. Nonspecific interactions were not observed when the slide was incubated with only the secondary antibody (not shown). Table 3 shows the peptides recognized by the anti-PspC3 serum. Interestingly, peptides involved in the binding of human FH (KLSRIKT) (46) and secretory immunoglobulin A (sIgA) (RNYPT and RNYPS) (47) were recognized.
PspC3 peptides recognized by sera from mice immunized with PspC3 plus Alum
DISCUSSION
We used peptide arrays to map the most immunogenic epitopes of different PspAs. Anti-PspA2 was the serum that recognized the lowest number of peptides, with these peptides mainly located at the nonconserved region among PspAs. In fact, our group previously showed that PspA2 elicited the least cross-protection of mice against an intranasal challenge with bacteria expressing heterologous PspAs (32). Anti-PspA3 serum recognized the largest number of peptides; however, most of them were peptides from the homologous sequence of PspA3.
We previously showed that anti-PspA4 and anti-PspA5 sera were broadly cross-reactive, recognizing pneumococci expressing PspA from clades 1 to 5 by Western blot analysis (31). Results from the peptide arrays pointed to the potential importance of the recognition of linear epitopes located in the N-terminal region (especially in the first third of this region) in the greater ability to recognize heterologous molecules by anti-PspA4 and anti-PspA5 sera. The importance of this more conserved region among PspAs has been described in the literature. Roche and collaborators (41) showed that the immunization of BALB/c mice with a fragment comprising amino acids 1 to 115 of PspA of the EF3296 strain (PspA clade 3) conferred protection to these animals against an intravenous challenge with EF3296. McDaniel and coworkers (44) also mapped some protective epitopes in regions covering the first 115 amino acids in the sequence of PspA from strain Rx1, using monoclonal antibodies raised against the same PspA.
In addition to the N-terminal region, some studies suggested the importance of the proline-rich region in the protection conferred after lethal challenge with pneumococcus. Brooks-Walter and collaborators (28) showed that immunization with PspC containing the proline-rich region protected mice challenged with strains of pneumococci that lacked the pspC gene. This cross-protection may be due to the reactivity of anti-PspC antibodies with the proline-rich region of PspA, since the proline regions in these two molecules are very similar. This observation was confirmed by the fact that anti-PspC antibodies lost the ability to bind to PspA after removal of the proline region in this molecule. Daniels and coworkers (48) showed that the proline blocks and the NonPro block (without prolines) within the proline-rich region were able to protect mice after intravenous challenge with pneumococci. In fact, it was possible in our study to observe the recognition of a peptide in the NonPro block within the proline-rich domain by anti-PspA4 serum in the peptide arrays. It may be that recognition of the NonPro block by anti-PspA4 antibodies potentiates the cross-recognition abilities of these antibodies.
Based on the results obtained with the peptide arrays, we decided to synthesize a gene encoding the N-terminal region of PspA. We used a synthetic gene encoding three repetitions of amino acids 6 to 44 of the mature PspA4 sequence, which is the PspA with the greatest cross-reactivity. The sera from mice immunized with the recombinant protein were able to recognize linear epitopes at the initial N-terminal region of different PspA clades in the peptide arrays and were capable of recognizing larger PspA molecules in ELISA. However, when tested by flow cytometry, the anti-PspAN-terminal-repeat antibodies were not able to bind to the surface of the two serotype 3 strains tested. Furthermore, the mice immunized with PspAN-terminal-repeat were not protected against a lethal intranasal challenge with ATCC 6303 (PspA clade 5). Since the N-terminal portion of PspA forms an antiparallel coiled-coil structure (49), this initial N-terminal region may be largely unexposed or camouflaged by the thick polysaccharide capsule present in the serotype 3 strains used in the flow cytometry and in the challenge experiments. Nevertheless, the experiments with a strain with a thinner serotype 14 capsule and with a nonencapsulated strain showing no binding of antibodies against PspAN-terminal-repeat ruled out this hypothesis. Another possibility is that the antibodies generated against linear epitopes do not bind efficiently to native PspA on the pneumococcal surface and are therefore not effective in protection. It is possible that conformational epitopes, which cannot be evaluated by these peptide arrays, are the determinants of protection mediated by anti-PspA antibodies.
In order to evaluate the interaction between anti-PspA antibodies and conformational epitopes of PspA, we decided to construct fragments containing 100 amino acids with a 50-amino-acid overlap covering the N-terminal region of the mature protein and the proline-rich region of PspA of strain Rx1. The capacities of the antibodies raised in mice against these fragments to bind onto the bacterial surface were tested by flow cytometry. Fragments 1, 2, 4, and 5 seemed to be exposed on the pneumococcal surface, while fragments 3, 6, and 7 did not seem to be exposed. Aiming at analyzing the protective capacities of these fragments, immunized mice were challenged with the A66.1 strain. In the groups immunized with the larger PspARx1 molecule, fragments 1, 2, 4, and 5 showed survival rates statistically different from that of the control group. This important result corroborates the result obtained in the flow cytometry assay, with the regions shown to be exposed on the bacterial surface by flow cytometry being the most protective in the challenge experiment. It is important to point out that PspA from strain Rx1 belongs to clade 2 and that the challenge strain used, A66.1, also expresses a clade 2 PspA. Fragments 4 and 5 provided 100% protection against the challenge, and the two of them comprised the clade-defining region (see Fig. S3 in the supplemental material), which was shown to be important in protection and to be the most variable region of PspA (13). Fragments 1 and 2 elicited partial protection (92% and 67%, respectively), but since these fragments are located at a more conserved part of the α-helical region of PspA, these regions may be important for inducing cross-protection against pneumococci expressing PspAs from different clades and families. Immunization with fragment 3 did not induce any enhancement in survival, which is in agreement with previous results showing that the central region of the α-helical region of PspA has a poor ability to induce even homologous protection (41). We did not observe protection in the mice immunized with the PspA fragments from Rx1 strain (PspA2) and challenged with the highly virulent ATCC 6303 strain (PspA5). Still, we think that this negative result does not rule out the importance of the more conserved N-terminal region of PspA. The use of more potent adjuvants or a mixture of fragments 1 and 2 may overcome this lack of cross-protection. Further mapping of PspA epitopes with monoclonal antibodies supports the hypothesis that conformational epitopes of PspA are crucial for the protective efficacy of this antigen (K. R. Genschmer and D. E. Briles, unpublished data). In prior studies (48), immunization with the proline-rich domain of PspA and inoculation with monoclonal antibody to the proline-rich domain were protective against intravenous challenge. The present results may indicate that immunity to the proline-rich domain is not as protective as immunity to the α-helical domains of PspA or that the antibody to the proline-rich domain, while protective against sepsis, cannot protect against pulmonary infection.
In contrast to the results obtained with PspA, antibodies to PspC showed recognition of important linear epitopes involved in the interaction with FH and sIgA. The 12-amino-acid motif responsible for binding to FH and its adjacent regions contain highly conserved residues among various PspC alleles (46). The conserved sIgA binding motif was found in all PspC proteins of groups 1 to 7 but not in serotype 3 strains expressing PspC8 or PspC11. PspC9 and PspC10 also do not have this motif, but these alleles were found as duplications in strains expressing alleles containing the sIgA binding motif (29). As this interaction between PspC and FH and/or sIgA is involved in the mechanisms of bacterial escape from the host immune system, obtaining antibodies that bind to these conserved regions responsible for the interaction of PspC with these molecules should play an important role against pneumococcal infections. Antibodies to a PspC from group 11 (PspC11) were shown in flow cytometry experiments to block the binding of FH to the homologous serotype 3 strain expressing PspC11 (50). Results published by our group also showed that preincubation with IgG anti-PspC3 led to partial inhibition of FH and sIgA binding to strains expressing different PspC variants in Western blot analyses and flow cytometry experiments (33).
In summary, we mapped immunogenic linear epitopes at the initial N-terminal region of PspA, but the induction of antibodies against these epitopes did not confer protection to mice against a lethal pneumococcal challenge. In contrast, the immunization of mice with 100-amino-acid fragments located at the N-terminal region of PspA induced protection against a pneumococcal challenge, indicating that the induction of antibodies against conformational epitopes present at the beginning of the N-terminal region of PspA may be an effective strategy for achieving cross-clade protection. Furthermore, recognition of the consensus sequences responsible for binding to FH and sIgA point to the importance of linear epitopes of PspC. Thus, mixtures of fragments or chimeric proteins containing protective epitopes of PspA and PspC, together with strong adjuvants, have the potential to be used as a vaccine that is capable of inducing broad protection against pneumococcal infections.
ACKNOWLEDGMENTS
We are grateful to Jorge M. C. Ferreira for the flow cytometry analysis.
This work was supported by FAPESP-MRC (Brazil-UK) (grant 2012/50816-4), Fundação Butantan (Brazil), and CNPq (Brazil) (grant 302070/2011-7). Cintia F. M. Vadesilho received a studentship from FAPESP (grant 2011/13671-5).
FOOTNOTES
- Received 14 April 2014.
- Returned for modification 24 April 2014.
- Accepted 28 April 2014.
- Accepted manuscript posted online 7 May 2014.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/CVI.00239-14.
- Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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