Mapping of epitopes recognized by antibodies induced by immunization of mice with PspA and PspC.

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.

Controlled human infection and rechallenge with Streptococcus pneumoniae reveals the protective efficacy of carriage in healthy adults.

RATIONALE: The immunological and protective role of pneumococcal carriage in healthy adults is not known, but high rates of disease and death in the elderly are associated with low carriage prevalence. OBJECTIVES: We employed an experimental human pneumococcal carriage model to investigate the immunizing effect of a single carriage episode. METHODS: Seventy healthy adults were challenged, and of those with carriage, 10 were rechallenged intranasally with live 6B Streptococcus pneumoniae up to 11 months after clearance of the first carriage episode. Serum and nasal wash antibody responses were measured before and after each challenge. MEASUREMENTS AND MAIN RESULTS: A total of 29 subjects were experimentally colonized. No subjects were colonized by experimental rechallenge, demonstrating the protective effect of initial carriage against subsequent infection. Carriage increased both mucosal and serum IgG levels to pneumococcal proteins and polysaccharide, resulting in a fourfold increase in opsonophagocytic activity. Importantly, passive transfer of postcarriage sera from colonized subjects conferred 70% protection against lethal challenge by a heterologous strain in a murine model of invasive pneumococcal pneumonia. These levels were significantly higher than the protection conferred by either precarriage sera (30%) or saline (10%). CONCLUSIONS: Experimental human carriage resulted in mucosal and systemic immunological responses that conferred protection against recolonization and invasive pneumococcal disease. These data suggest that mucosal pneumococcal vaccination strategies may be important for vulnerable patient groups, particularly the elderly, who do not sustain carriage.

Characterization of the antibody response elicited by immunization with pneumococcal surface protein A (PspA) as recombinant protein or DNA vaccine and analysis of protection against an intranasal lethal challenge with Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) is an important candidate for a vaccine against pneumococcal infections. DNA vaccines expressing PspA were shown to protect mice against intraperitoneal and colonization challenge models in mice. We now show that a DNA vaccine expressing PspA from clade 4 (pSec-pspA4Pro) is also able to elicit protection against an intranasal lethal challenge model at levels similar to the recombinant protein PspA4Pro adjuvanted with alum. PspA4Pro + alum induced an IgG response characterized by a high IgG1/IgG2a ratio, leading to a lack of binding of anti-PspA IgG2a antibodies to intact pneumococci in vitro, which is in contrast to the response elicited by pSec-pspA4Pro. Epitopes recognized by the sera were mapped and antibodies induced by immunization with PspA4Pro + alum showed positive reaction with several synthetic peptides, mostly located in the first half of the protein. On the other hand, antibodies induced by the DNA vaccine showed reactivity with only two peptides. Though both strategies were protective against the intranasal lethal challenge model, the elicited humoral responses differ significantly, with the detection of important differences in the Fc (IgG1/IgG2a ratios) and Fab (recognized epitopes) regions of the induced antibodies.

Controlled inflammatory responses in the lungs are associated with protection elicited by a pneumococcal surface protein A-based vaccine against a lethal respiratory challenge with Streptococcus pneumoniae in mice.

Streptococcus pneumoniae is a pathogen of great importance worldwide. We have previously described the efficacy of a nasal vaccine composed of the pneumococcal surface protein A and the whole-cell pertussis vaccine as an adjuvant against a pneumococcal invasive challenge in mice. Spread of bacteria to the bloodstream was probably prevented by the high levels of systemic antibodies induced by the vaccine, but bacteria were only cleared from the lungs 3 weeks later, indicating that local immune responses may contribute to survival. Here we show that a strict control of inflammatory responses in lungs of vaccinated mice occurs even in the presence of high numbers of pneumococci. This response was characterized by a sharp peak of neutrophils and lymphocytes with a simultaneous decrease in macrophages in the respiratory mucosa at 12 h postchallenge. Secretion of interleukin-6 (IL-6) and gamma interferon (IFN-γ) was reduced at 24 h postchallenge, and the induction of tumor necrosis factor alpha (TNF-α) secretion, observed in the first hours postchallenge, was completely abolished at 24 h. Before challenge and at 12 h postchallenge, vaccinated mice displayed higher numbers of CD4(+) T, CD8(+) T, and B lymphocytes in the lungs. However, protection still occurs in the absence of each of these cells during the challenge, indicating that other effectors may be related to the prevention of lung injuries in this model. High levels of mucosal anti-PspA antibodies were maintained in vaccinated mice during the challenge, suggesting an important role in protection.

Cross-reactivity of antipneumococcal surface protein C (PspC) antibodies with different strains and evaluation of inhibition of human complement factor H and secretory IgA binding via PspC.

Pneumococcal surface protein C (PspC) is an important candidate for a cost-effective vaccine with broad coverage against pneumococcal diseases. Previous studies have shown that Streptococcus pneumoniae is able to bind to both human factor H (FH), an inhibitor of complement alternative pathway, and human secretory IgA (sIgA) via PspC. PspC was classified into 11 groups based on variations of the gene. In this work, we used three PspC fragments from different groups (PspC3, PspC5, and PspC8) to immunize mice for the production of antibodies. Immunization with PspC3 induced antibodies that recognized the majority of the clinical isolates as analyzed by Western blotting of whole-cell extracts and flow cytometry of intact bacteria, while anti-PspC5 antibodies showed cross-reactivity with the paralogue pneumococcal surface protein A (PspA), and anti-PspC8 antibodies reacted only with the PspC8-expressing strain. Most of the isolates tested showed strong binding to FH and weaker interaction with sIgA. Preincubation with anti-PspC3 and anti-PspC5 IgG led to some inhibition of binding of FH, and preincubation with anti-PspC3 partially inhibited sIgA binding in Western blotting. The analysis of intact bacteria through flow cytometry showed only a small decrease in FH binding after incubation of strain D39 with anti-PspC3 IgG, and one clinical isolate showed inhibition of sIgA binding by anti-PspC3 IgG. We conclude that although anti-PspC3 antibodies were able to recognize PspC variants from the majority of the strains tested, partial inhibition of FH and sIgA binding through anti-PspC3 antibodies in vitro could be observed for only a restricted number of isolates.

Combination of pneumococcal surface protein A (PspA) with whole cell pertussis vaccine increases protection against pneumococcal challenge in mice.

Streptococcus pneumoniae is the leading cause of respiratory acute infections around the world. In Latin America, approximately 20,000 children under 5 years of age die of pneumococcal diseases annually. Pneumococcal surface protein A (PspA) is among the best-characterized pneumococcal antigens that confer protection in animal models of pneumococcal infections and, as such, is a good alternative for the currently available conjugated vaccines. Efficient immune responses directed to PspA in animal models have already been described. Nevertheless, few low cost adjuvants for a subunit pneumococcal vaccine have been proposed to date. Here, we have tested the adjuvant properties of the whole cell Bordetella pertussis vaccine (wP) that is currently part of the DTP (diphtheria-tetanus-pertussis) vaccine administrated to children in several countries, as an adjuvant to PspA. Nasal immunization of BALB/c mice with a combination of PspA5 and wP or wP(low)--a new generation vaccine that contains low levels of B. pertussis LPS--conferred protection against a respiratory lethal challenge with S. pneumoniae. Both PspA5-wP and PspA5-wP(low) vaccines induced high levels of systemic and mucosal antibodies against PspA5, with similar profile, indicating no essential requirement for B. pertussis LPS in the adjuvant properties of wP. Accordingly, nasal immunization of C3H/HeJ mice with PspA5-wP conferred protection against the pneumococcal challenge, thus ruling out a role for TLR4 responses in the adjuvant activity and the protection mechanisms triggered by the vaccines. The high levels of anti-PspA5 antibodies correlated with increased cross-reactivity against PspAs from different clades and also reflected in cross-protection. In addition, passive immunization experiments indicated that antibodies played an important role in protection in this model. Finally, subcutaneous immunization with a combination of PspA5 with DTP(low) protected mice against challenge with two different pneumococcal strains, opening the possibility for the development of a combined infant vaccine composed of DTP and PspA.

Protection against nasal colonization with Streptococcus pneumoniae by parenteral immunization with a DNA vaccine encoding PspA (Pneumococcal surface protein A).

Pneumococcal surface protein A (PspA) is an important candidate for a cost-effective vaccine with broad coverage against Streptococcus pneumoniae. We have previously shown that intramuscular immunization with PspA as a DNA vaccine induces an immune response characterized by the induction of a balanced IgG1/IgG2a antibody response in BALB/c mice, which was able to efficiently mediate complement deposition onto intact bacteria and to induce protection against an intraperitoneal challenge. We now confirm the results in C57BL/6 mice and further show that the response induced by the DNA vaccine expressing PspA is able to mediate protection against colonization of the nasopharyngeal mucosa even though immunization was given parenterally. Moreover, a positive correlation was observed between IgG1 and the numbers of CFU recovered, whereas an inverse correlation was observed between nasal CFU levels and IgG2a. A positive correlation was also found for IgG1/IgG2a antibody ratios with CFU recovered from the nasopharynx. Therefore, reduction of nasal colonization was strongly associated with increased levels of serum IgG2a complement fixing antibody and low levels of IgG1 antibody which has much less complement fixing activity. Passive transfer of serum from animals immunized with the DNA vaccine expressing PspA was also able to reduce the fraction of mice with high density of colonization of the nasopharynx. Secretion of IFN-gamma, but not IL-17, was observed in splenocytes from mice immunized with the DNA vaccine.

Immunization of mice with single PspA fragments induces antibodies capable of mediating complement deposition on different pneumococcal strains and cross-protection.

PspA is an important candidate for a vaccine with serotype-independent immunity against pneumococcal infections. Based on sequence relatedness, PspA has been classified into three families comprising six clades. We have previously addressed the cross-reactivity of antibodies against PspA fragments containing the N-terminal and proline-rich regions of PspA from clades 1 to 5 (PspA1, PspA2, PspA3, PspA4, and PspA5) by Western blot analysis and reported that anti-PspA4 and anti-PspA5 were able to recognize pneumococci expressing PspA proteins from all of the clades analyzed. We have now analyzed the functional capacity of these antibodies to bind and to mediate complement deposition on intact bacteria in vitro. Our results show that both PspA4 and PspA5 elicit antibodies that are able to bind and to mediate complement deposition efficiently on pneumococcal strains bearing PspA proteins from clades 1 to 5. Moreover, mice immunized with PspA4 and PspA5 were protected against an intranasal lethal challenge with strains expressing PspA proteins from the two major families. PspA4 and PspA5 are thus able to induce antibodies with a high degree of cross-reactivity in vitro, which is reflected in cross-protection of mice. We have also analyzed the contribution of the nonproline (NonPro) block within the conserved proline-rich region to the reactivity of anti-PspA antibodies, and the results indicate that N-terminal alpha-helical region, the blocks of proline repeats, and the NonPro region can influence the degree of cross-reactivity of antibodies to PspA.

Combination of Pneumococcal Surface Protein A (PspA)with Whole Cell Pertussis Vaccine Increases ProtectionAgainst Pneumococcal Challenge in Mice

Streptococcus pneumoniae is the leading cause of respiratory acute infections around the world. In Latin America, approximately 20,000 children under 5 years of age die of pneumococcal diseases annually. Pneumococcal surface protein PspA) is among the best-characterized pneumococcal antigens that confer protection in animal models of pneumococcal infections and, as such, is a good alternative for the currently available conjugated vaccines. Efficient immune responses directed to PspA in animal models have already been described. Nevertheless, few low cost adjuvants for a subunit pneumococcal vaccine have been proposed to date. Here, we have tested the adjuvant properties of the whole cell Bordetella pertussis vaccine (wP) that is currently part of the DTP (diphtheria-tetanus-pertussis) vaccine administrated to children in several countries, as an adjuvant to PspA. Nasal immunization of BALB/c mice with a combination of PspA5 and wP or wPlow – a new generation vaccine that contains low levels of B. pertussis LPS – conferred protection against a respiratory lethal challenge with S. pneumoniae. Both PspA5-wP and PspA5-wPlow vaccines induced high levels of systemic and mucosal antibodies against PspA5, with similar profile, indicating no essential requirement for B...

Comparison of the pulmonary response against lethal and non-lethal intranasal challenges with two different pneumococcal strains.

The differences between the immune response elicited during a self-limiting and a life-threatening lung infection with Streptococcus pneumoniae was analyzed in a mouse model of intranasal challenge using two different pneumococcal strains. M10, a serotype 11A strain, induced an early response within the first 12h after the challenge, which was characterized by the early local secretion of TNF-alpha and IL-6, followed by a sharp and rapid neutrophil influx. Bacterial loads in the lungs already started to fall at 12h after the challenge and no pneumococci could be recovered after 36h, at the time point when the animals started to show improvement in disease symptoms. ATCC6303, a serotype 3 strain, on the other hand, showed only a late increase in local TNF-alpha and IL-6 levels, when bacterial growth already seems to be out of control. Although cell influx was also observed, neutrophil rise was not as marked as with M10 (type 11A). Pneumococcal loads increased constantly and bacteria started to be recovered from the blood at 30h after the challenge. After this time point, animals showed worsening of symptoms and became lethargic. The resolution of the acute infection could be thus correlated with the early induction of proinflammatory cytokines, which could be due to the presence of a thinner polysaccharide capsule in M10 (type 11A), rendering bacterial components capable of activating the innate immune response more accessible.

Recognition of pneumococcal isolates by antisera raised against PspA fragments from different clades.

Pneumococcal surface protein A (PspA) is an important vaccine candidate against pneumococcal infections, capable of inducing protection in different animal models. Based on its structural diversity, it has been suggested that a PspA-based vaccine should contain at least one fragment from each of the two major families (family 1, comprising clades 1 and 2, and family 2, comprising clades 3, 4 and 5) in order to elicit broad protection. This study analysed the recognition of a panel of 35 pneumococcal isolates bearing different PspAs by antisera raised against the N-terminal regions of PspA clades 1 to 5. The antiserum to PspA clade 4 was found to show the broadest cross-reactivity, being able to recognize pneumococcal strains containing PspAs of all clades in both families. The cross-reactivity of antibodies elicited against a PspA hybrid including the N-terminal region of clade 1 fused to a shorter and more divergent fragment (clade-defining region, or CDR) of clade 4 (PspA1-4) was also tested, and revealed a strong recognition of isolates containing clades 1, 4 and 5, and weaker reactions with clades 2 and 3. The analysis of serum reactivity against different PspA regions further revealed that the complete N-terminal region rather than just the CDR should be included in an anti-pneumococcal vaccine. A PspA-based vaccine is thus proposed to be composed of the whole N-terminal region of clades 1 and 4, which could also be expressed as a hybrid protein.