Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge

Strategies for the development of new vaccines against Streptococcus pneumoniae infections try to overcome problems such as serotype coverage and high costs, present in currently available vaccines. Formulations based on protein candidates that can induce protection in animal models have been pointed as good alternatives. Among them, the Pneumococcal Surface Protein A (PspA) plays an important role during systemic infection at least in part through the inhibition of complement deposition on the pneumococcal surface, a mechanism of evasion from the immune system. Antigen delivery systems based on live recombinant lactic acid bacteria (LAB) represents a promising strategy for mucosal vaccination, since they are generally regarded as safe bacteria able to elicit both systemic and mucosal immune responses. In this work, the N-terminal region of clade 1 PspA was constitutively expressed in Lactobacillus casei and the recombinant bacteria was tested as a mucosal vaccine in mice. Nasal immunization with L. casei-PspA 1 induced anti-PspA antibodies that were able to bind to pneumococcal strains carrying both clade 1 and clade 2 PspAs and to induce complement deposition on the surface of the bacteria. In addition, an increase in survival of immunized mice after a systemic challenge with a virulent pneumococcal strain was observed.     

Characterization of selected strains of pneumococcal surface protein A.

Several proteins, in addition to the polysaccharide capsule, have recently been implicated in the full virulence of the Streptococcus pneumoniae bacterial pathogen. One of these novel virulence factors of S. pneumoniae is pneumococcal surface protein A (PspA). The N-terminal, cell surface exposed, and functional part of PspA is essential for full pneumococcal virulence, as evidenced by the fact that antibodies raised against this part of the protein are protective against pneumococcal infections. PspA has recently been implicated in anti-complementary function as it reduces complement-mediated clearance and phagocytosis of pneumococci. Several recombinant N-terminal fragments of PspA from different strains of pneumococci, Rx1, BG9739, BG6380, EF3296, and EF5668, were analyzed using circular dichroism, analytical ultracentrifugation sedimentation velocity and equilibrium methods, and sequence homology. Uniformly, all strains of PspA molecules studied have a high alpha-helical secondary structure content and they adopt predominantly a coiled-coil structure with an elongated, likely rod-like shape. No beta-sheet structures were detected for any of the PspA molecules analyzed. All PspAs were found to be monomeric in solution with the exception of the BG9739 strain which had the propensity to partially aggregate but only into a tetrameric form. These structural properties were correlated with the functional, anti-complementary properties of PspA molecules based on the polar distribution of highly charged termini of its coiled-coil domain. The recombinant Rx1 PspA is currently under consideration for pneumococcal vaccine development.     

Maternal immunization with pneumococcal surface protein A protects against pneumococcal infections among derived offspring

Pathogen-specific antibody plays an important role in protection against pneumococcal carriage and infections. However, neonates and infants exhibit impaired innate and adaptive immune responses, which result in their high susceptibility to pneumococci. To protect neonates and infants against pneumococcal infection it is important to elicit specific protective immune responses at very young ages. In this study, we investigated the protective immunity against pneumococcal carriage, pneumonia, and sepsis induced by maternal immunization with pneumococcal surface protein A (PspA). Mother mice were intranasally immunized with recombinant PspA (rPspA) and cholera toxin B subunit (CTB) prior to being mated. Anti-PspA specific IgG, predominantly IgG1, was present at a high level in the serum and milk of immunized mothers and in the sera of their pups. The pneumococcal densities in washed nasal tissues and in lung homogenate were significantly reduced in pups delivered from and/or breast-fed by PspA-immunized mothers. Survival after fatal systemic infections with various types of pneumococci was significantly extended in the pups, which had received anti-PspA antibody via the placenta or through their milk. The current findings strongly suggest that maternal immunization with PspA is an attractive strategy against pneumococcal infections during early childhood.     

PspA Family Distribution,Antimicrobial Resistance and Serotype of Streptococcus pneumoniae Isolated from Upper Respiratory Tract Infections in Japan

Background

The protection against pneumococcal infections provided by currently available pneumococcal polysaccharide conjugate vaccines are restricted to the limited number of the serotypes included in the vaccine. In the present study, we evaluated the distribution of the pneumococcal capsular type and surface protein A (PspA) family of pneumococcal isolates from upper respiratory tract infections in Japan.

Methods

A total of 251 S. pneumoniae isolates from patients seeking treatment for upper respiratory tract infections were characterized for PspA family, antibiotic resistance and capsular type.

Results

Among the 251 pneumococci studied, the majority (49.4%) was identified as belonging to PspA family 2, while most of the remaining isolates (44.6%) belonged to family 1. There were no significant differences between the distributions of PspA1 versus PspA2 isolates based on the age or gender of the patient, source of the isolates or the isolates’ susceptibilities to penicillin G. In contrast, the frequency of the mefA gene presence and of serotypes 15B and 19F were statistically more common among PspA2 strains.

Conclusion

The vast majority of pneumococci isolated from the middle ear fluids, nasal discharges/sinus aspirates or pharyngeal secretions represented PspA families 1 and 2. Capsular serotypes were generally not exclusively associated with certain PspA families, although some capsular types showed a much higher proportion of either PspA1 or PspA2. A PspA-containing vaccine would potentially provide high coverage against pneumococcal infectious diseases because it would be cross-protective versus invasive disease with the majority of pneumococci infecting children and adults.     

Protective efficacy of PspA (pneumococcal surface protein A)-based DNA vaccines: contribution of both humoral and cellular immune responses

Streptococcus pneumoniae is a major public health problem and new strategies for the development of cost-effective alternative vaccines are important. The use of protein antigens such as PspA (pneumococcal surface protein A) is a promising approach to increase coverage at reduced costs. We have previously described the induction of a strong antibody response by a DNA vaccine expressing a C-terminal fragment of PspA. Fusion of this fragment with the cytoplasmic variant of SV40 large T-antigen (CT-Ag) caused reduction in specific interferon-gamma produced by stimulated spleen cells. In this work we show that the DNA vaccine expressing the C-terminal region of PspA elicits significant protection in mice against intraperitoneal challenge with a virulent strain of S. pneumoniae. Furthermore, fusion with CT-Ag completely abrogated the protection elicited by DNA immunization with this fragment. In this case, protection did not correlate with total anti-PspA antibody production nor with total IgG2a levels. The anti-PspA sera obtained from both constructs showed equivalent opsonic activity of pneumococci, indicating that the antibodies produced were functional. We could, though, observe a correlation between a lower IgG1:IgG2a ratio, which is indicative of a stronger bias towards Th1 responses, and protection. We also show that a vector expressing the most variable N-terminal alpha-helical region induces higher antibody formation, with increased protection of mice against intraperitoneal challenge with a more virulent strain of S. pneumoniae. As a whole, these results indicate that antibodies elicited against PspA would not be solely responsible for the protection induced by DNA vaccination and that cell-mediated immune responses could also be involved in protection against pneumococcal sepsis.     

Identification of glycosylated regions in pneumococcal PspA conjugated to serotype 6B capsular polysaccharide

Conjugate vaccines are being widely used since their introduction. Nowadays the interest in these vaccines is still growing and new antigens and conjugate chemistry are being studied and developed. Pneumococcal surface protein A (PspA) is one of the most studied pneumococcal antigens and is an important vaccine candidate. One approach to broaden the conjugate vaccine coverage could be the conjugation of the polysaccharide to a pneumococcal protein such as PspA. Previous results have shown that conjugated recombinant fragment of PspA (rPspA) not only maintained but also in some conjugates improved the induction of protective antibodies raised against the protein carrier. We describe here a characterization study to identify the domains of Streptococcus pneumoniae recombinant PspA (rPspA), from family 1 clade 1 and family 2 clade 3, involved in the conjugation with serotype 6B capsular polysaccharide.     

Conserved Surface Accessible Nucleoside ABC Transporter Component SP0845 Is Essential for Pneumococcal Virulence and Confers Protection In Vivo

Streptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis and meningitis. Surface accessible proteins of S. pneumoniae are being explored for the development of a protein-based vaccine in order to overcome the limitations of existing polysaccharide-based pneumococcal vaccines. To identify a potential vaccine candidate, we resolved surface-associated proteins of S. pneumoniae TIGR4 strain using two-dimensional gel electrophoresis followed by immunoblotting with antisera generated against whole heat-killed TIGR4. Ten immunoreactive spots were identified by mass spectrometric analysis that included a putative lipoprotein SP0845. Analysis of the inferred amino acid sequence of sp0845 homologues from 36 pneumococcal strains indicated that SP0845 was highly conserved (>98% identity) and showed less than 11% identity with any human protein. Our bioinformatic and functional analyses demonstrated that SP0845 is the substrate-binding protein of an ATP-binding cassette (ABC) transporter that is involved in nucleoside uptake with cytidine, uridine, guanosine and inosine as the preferred substrates. Deletion of the gene encoding SP0845 renders pneumococci avirulent suggesting that it is essential for virulence. Immunoblot analysis suggested that SP0845 is expressed in in vitro grown pneumococci and during mice infection. Immunofluorescence microscopy and flow cytometry data indicated that SP0845 is surface exposed in encapsulated strains and accessible to antibodies. Subcutaneous immunization with recombinant SP0845 induced high titer antibodies in mice. Hyperimmune sera raised against SP0845 promoted killing of encapsulated pneumococcal strains in a blood bactericidal assay. Immunization with SP0845 protected mice from intraperitoneal challenge with heterologous pneumococcal serotypes. Based on its surface accessibility, role in virulence and ability to elicit protective immunity, we propose that SP0845 may be a potential candidate for a protein-based pneumococcal vaccine.     

Diversity of pneumococcal surface protein A (PspA) among prevalent clones in Spain

Background  

PspA is recognized as a major pneumococcal virulence factor and a possible vaccine candidate. The aim of this study was to analyze the PspA family and clade distribution among 112 Spanish pneumococci representatives of dominant clones among patients with invasive disease (n = 66) and nasopharyngeal healthy carriage in children (n = 46).     

The genetic background of Streptococcus pneumoniae affects protection in mice immunized with PspA

Anti-PspA antibodies are less efficient at protecting mice against certain pneumococcal strains. Immunization with PspA from EF5668 provided better protection against WU2 (a different capsular serotype and PspA family) than against EF5668. To understand the role of the pneumococcal genetic background in anti-PspA-mediated protection, we constructed a mutant of WU2 expressing pspA from EF5668. Both passive and active immunization demonstrated that the genetic background impacted the protection mediated by anti-PspA antibodies. We localized the protection-eliciting region to the first 122 amino acid residues of the N-terminus of the alpha-helical domain of PspA/EF5668.     

Interleukin-17A mediates acquired immunity to pneumococcal colonization

Although anticapsular antibodies confer serotype-specific immunity to pneumococci, children increase their ability to clear colonization before these antibodies appear, suggesting involvement of other mechanisms. We previously reported that intranasal immunization of mice with pneumococci confers CD4+ T cell-dependent, antibody- and serotype-independent protection against colonization. Here we show that this immunity, rather than preventing initiation of carriage, accelerates clearance over several days, accompanied by neutrophilic infiltration of the nasopharyngeal mucosa. Adoptive transfer of immune CD4+ T cells was sufficient to confer immunity to na?ve RAG1(-/-) mice. A critical role of interleukin (IL)-17A was demonstrated: mice lacking interferon-gamma or IL-4 were protected, but not mice lacking IL-17A receptor or mice with neutrophil depletion. In vitro expression of IL-17A in response to pneumococci was assayed: lymphoid tissue from vaccinated mice expressed significantly more IL-17A than controls, and IL-17A expression from peripheral blood samples from immunized mice predicted protection in vivo. IL-17A was elicited by pneumococcal stimulation of tonsillar cells of children or adult blood but not cord blood. IL-17A increased pneumococcal killing by human neutrophils both in the absence and in the presence of antibodies and complement. We conclude that IL-17A mediates pneumococcal immunity in mice and probably in humans; its elicitation in vitro could help in the development of candidate pneumococcal vaccines.     

Passive protection against lethal enterovirus 71 infection in newborn mice by neutralizing antibodies elicited by a synthetic peptide

Enterovirus 71 (EV71) infections could lead to high mortalities and neither vaccine nor therapeutic treatment is available. We investigated vaccination with a synthetic peptide SP70 representing a neutralizing linear VP1 epitope of EV71 strain 41 (subgenogroup B4) and passive transfer of anti-SP70 antibodies to protect suckling Balb/c mice against EV71 infectivity. When the mouse anti-SP70 antisera with a neutralizing antibody titer of 1:32 were passively administered to one-day-old suckling mice which had been challenged with a lethal dose of 1000 TCID(50) per mouse, the neutralizing anti-SP70 antibodies were able to confer 80% in vivo protection. In contrast, suckling mice which did not receive any anti-SP70 antisera did not survive the viral challenge at day 21 postinfection. Histological examination and real-time RT-PCR assays revealed viral infiltration in small intestines of EV71-infected mice. Interestingly, anti-SP70 antibodies play a major role in the inhibition of EV71 replication in vivo and significantly reduced the viral titer. In conclusion, EV71-neutralizing antibodies elicited by the synthetic peptide SP70 were able to confer good in vivo passive protection against homologous and heterologous EV71 strains in suckling Balb/c mice.     

The virulence function of Streptococcus pneumoniae surface protein A involves inhibition of complement activation and impairment of complement receptor-mediated protection

Complement is important for elimination of invasive microbes from the host, an action achieved largely through interaction of complement-decorated pathogens with various complement receptors (CR) on phagocytes. Pneumococcal surface protein A (PspA) has been shown to interfere with complement deposition onto pneumococci, but to date the impact of PspA on CR-mediated host defense is unknown. To gauge the contribution of CRs to host defense against pneumococci and to decipher the impact of PspA on CR-dependent host defense, wild-type C57BL/6J mice and mutant mice lacking CR types 1 and 2 (CR1/2(-/-)), CR3 (CR3(-/-)), or CR4 (CR4(-/-)) were challenged with WU2, a PspA(+) capsular serotype 3 pneumococcus, and its PspA(-) mutant JY1119. Pneumococci also were used to challenge factor D-deficient (FD(-/-)), LFA-1-deficient (LFA-1(-/-)), and CD18-deficient (CD18(-/-)) mice. We found that FD(-/-), CR3(-/-), and CR4(-/-) mice had significantly decreased longevity and survival rate upon infection with WU2. In comparison, PspA(-) pneumococci were virulent only in FD(-/-) and CR1/2(-/-) mice. Normal mouse serum supported more C3 deposition on pneumococci than FD(-/-) serum, and more iC3b was deposited onto the PspA(-) than the PspA(+) strain. The combined results confirm earlier conclusions that the alternative pathway of complement activation is indispensable for innate immunity against pneumococcal infection and that PspA interferes with the protective role of the alternative pathway. Our new results suggest that complement receptors CR1/2, CR3, and CR4 all play important roles in host defense against pneumococcal infection.     

Purification and characterization of macrolide 2''-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics

Hyperimmune and high-titered polyclonal pneumococcal antisera, specific for cross-reactive types within groups, were produced in adult rabbits. Purified capsular polysaccharide was injected intravenously into adult rabbits. One week later, these rabbits were given multiple intravenous injections of formalin-inactivated pneumococci of the cross-reactive type by an established method. Each of the resultant antisera were specific for the cross-reactive type indicating that the previous injection of the polysaccharide had induced epitope-specific tolerance. This method was successful for production of antisera against pneumococcal types 6A, 6B, 9N, 9V, 19F and 19A. Polyclonal rabbit pneumococcal antisera have some advantages over murine monoclonal antibodies for serologic studies and this method should be applicable for producing type-specific antibodies to cross-reactive polysaccharides of clinical interest. Further, this method is simpler and generally produces higher titered monovalent (factor) reagents than absorbed antisera.     

Diversity of Pneumolysin and Pneumococcal Histidine Triad Protein D of Streptococcus pneumoniae Isolated from Invasive Diseases in Korean Children

Pneumolysin (Ply) and pneumococcal histidine triad protein D (PhtD) are candidate proteins for a next-generation pneumococcal vaccine. We aimed to analyze the genetic diversity and antigenic heterogeneity of Ply and PhtD for 173 pneumococci isolated from invasive diseases in Korean children. Allele was designated based on the variation of amino acid sequence. Antigenicity was predicted by the amino acid hydrophobicity of the region. There were seven and 39 allele types for the ply and phtD genes, respectively. The nucleotide sequence identity was 97.2%-99.9% for ply and 91.4%-98.0% for phtD gene. Only minor variations in hydrophobicity were noted among the antigenicity plots of Ply and PhtD. Overall, the allele types of the ply and phtD genes were remarkably homogeneous, and the antigenic diversity of the corresponding proteins was very limited. The Ply and PhtD could be useful antigens for universal pneumococcal vaccines.     

Contribution of novel choline-binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae

The surface of Streptococcus pneumoniae is decorated with a family of choline-binding proteins (CBPs) that are non-covalently bound to the phosphorylcholine of the teichoic acid. Two examples (PspA, a protective antigen, and LytA, the major autolysin) have been well characterized. We identified additional CPBs and characterized a new CBP, CbpA, as an adhesin and a determinant of virulence. Using choline immobilized on a solid matrix, a mixture of proteins from a pspA -deficient strain of pneumococcus was eluted in a choline-dependent fashion. Antisera to these proteins passively protected mice challenged in the peritoneum with a lethal dose of pneumococci. The predominant component of this mixture, CbpA, is a 75-kDa surface-exposed protein that reacts with human convalescent antisera. The deduced sequence from the corresponding gene showed a chimeric architecture with a unique N-terminal region and a C-terminal domain consisting of 10 repeated choline-binding domains nearly identical to PspA. A cbpA -deficient mutant showed a >50% reduction in adherence to cytokine-activated human cells and failed to bind to immobilized sialic acid or lacto-N-neotetraose, known pneumococcal ligands on eukaryotic cells. Carriage of this mutant in an animal model of nasopharyngeal colonization was reduced 100-fold. There was no difference between the parent strain and this mutant in an intraperitoneal model of sepsis. These data for CbpA extend the important functions of the CBP family to bacterial adherence and identify a pneumococcal vaccine candidate.     

Proteins of Streptococcus pneumoniae: perspectives for development of pneumococcal vaccine

Streptococcus pneumoniae cell wall and cytoplasmic proteins contribute directly to pathogenesis of pneumococcal infection. Protective effect of pneumococcal proteins such as pneumolysin (Ply), muramylamidase (LytA) and pneumococcal surface protein A (PspA). There is discussion in the literature about development of conjugared pneumococcal vaccines, which should include polysaccharides of invasive serotypes of pneumococci as well as protein antigens of this pathogen, for prevention of infections caused by S. pneumoniae. Researches suggest that such hybrid vaccines will be effective, first of all, for children < 2 years of age and elderly > 65 years old because immune response to polysaccharide vaccines either do not form at all or insufficient for prevention of pneumococcal infection.     

Helper T Cell Epitope-Mapping Reveals MHC-Peptide Binding Affinities That Correlate with T Helper Cell Responses to Pneumococcal Surface Protein A

Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Several proteins and polysaccharide capsule have recently been implicated in the virulence of and protective immunity against Streptococcus pneumonia. Pneumococcal surface protein A (PspA) is highly conserved among S. pneumonia strains, inhibits complement activation, binds lactoferrin, elicits protective systemic immunity against pneumococcal infection, and is necessary for full pneumococcal virulence. Identification of PspA peptides that optimally bind human leukocyte antigen (HLA) would greatly contribute to global vaccine efforts, but this is hindered by the multitude of HLA polymorphisms. Here, we have used an experimental data set of 54 PspA peptides and in silico methods to predict peptide binding to HLA and murine major histocompatibility complex (MHC) class II. We also characterized spleen- and cervical lymph node (CLN)-derived helper T lymphocyte (HTL) cytokine responses to these peptides after S. pneumonia strain EF3030-challenge in mice. Individual, yet overlapping peptides, 15 amino acids in length revealed residues 199 to 246 of PspA (PspA_199–246) consistently caused the greatest IFN-γ, IL-2, IL-5 and proliferation as well as moderate IL-10 and IL-4 responses by ex vivo stimulated splenic and CLN CD4⁺ T cells isolated from S. pneumonia strain EF3030-challeged F₁ (B6×BALB/c) mice. IEDB, RANKPEP, SVMHC, MHCPred, and SYFPEITHI in silico analysis tools revealed peptides in PspA_199–246 also interact with a broad range of HLA-DR, -DQ, and -DP allelles. These data suggest that predicted MHC class II-peptide binding affinities do not always correlate with T helper (Th) cytokine or proliferative responses to PspA peptides, but when used together with in vivo validation can be a useful tool to choose candidate pneumococcal HTL epitopes.     

Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway

Overexpression of either heterologous or homologous proteins that are routed to the periplasm via the twin-arginine translocation (Tat) pathway results in a block of export and concomitant accumulation of the respective protein precursor in the cytoplasm. Screening of a plasmid-encoded genomic library for mutants that confer enhanced export of a TorA signal sequence (ssTorA)-GFP-SsrA fusion protein, and thus result in higher cell fluorescence, yielded the pspA gene encoding phage shock protein A. Coexpression of pspA relieved the secretion block observed with ssTorA-GFP-SsrA or upon overexpression of the native Tat proteins SufI and CueO. A similar effect was observed with the Synechocystis sp. strain PCC6803 PspA homologue, VIPP1, indicating that the role of PspA in Tat export may be phylogenetically conserved. Mutations in Tat components that completely abolish export result in a marked induction of PspA protein synthesis, consistent with its proposed role in enhancing protein translocation via Tat.     

Cefditoren and Ceftriaxone Enhance Complement-Mediated Immunity in the Presence of Specific Antibodies against Antibiotic-Resistant Pneumococcal Strains

Background

Specific antibodies mediate humoral and cellular protection against invading pathogens such as Streptococcus pneumoniae by activating complement mediated immunity, promoting phagocytosis and stimulating bacterial clearance. The emergence of pneumococcal strains with high levels of antibiotic resistance is of great concern worldwide and a serious threat for public health.

Methodology/Principal Findings

Flow cytometry was used to determine whether complement-mediated immunity against three antibiotic-resistant S. pneumoniae clinical isolates is enhanced in the presence of sub-inhibitory concentrations of cefditoren and ceftriaxone. The binding of acute phase proteins such as C-reactive protein and serum amyloid P component, and of complement component C1q, to pneumococci was enhanced in the presence of serum plus either of these antibiotics. Both antibiotics therefore trigger the activation of the classical complement pathway against S. pneumoniae. C3b deposition was also increased in the presence of specific anti-pneumococcal antibodies and sub-inhibitory concentrations of cefditoren and ceftriaxone confirming that the presence of these antibiotics enhances complement-mediated immunity to S. pneumoniae.

Conclusions/Significance

Using cefditoren and ceftriaxone to promote the binding of acute phase proteins and C1q to pneumococci, and to increase C3b deposition, when anti-pneumococcal antibodies are present, might help reduce the impact of antibiotic resistance in S. pneumoniae infections.     

Epitope analyses of pneumococcal surface protein A: a combination of two monoclonal antibodies detects 94% of clinical isolates

Immunisation of BALB/c mice with seven heat-treated Norwegian clinical isolates of Streptococcus pneumoniae of different serotypes elicited mainly monoclonal antibodies (mAbs) to pneumococcal surface protein A (PspA). It was remarkable that the fusions resulted only in a few mAbs directed against other protein antigens. Dot blot analysis with 16 mAbs using clinical isolates representing 23 different capsular types and the uncapsulated reference strain R36A showed that some of the mAbs bound to PspA epitopes expressed by a low number of strains whereas others bound to broadly distributed epitopes. On the basis of their reactivities, seven of these mAbs could be divided into two groups recognising different subsets of pneumococci. The three mAbs in the narrow reacting group bound to epitopes found in 21-25% of the strains whereas the four mAbs in the broad reacting group detected more than 57% of the analysed strains. The epitopes for these seven antibodies were surface exposed on live exponential phase grown pneumococci as shown by flow cytometry. The finding that a combination of mAb 180,C-1 (IgG2a) from the first group and mAb 170,E-11 (IgG2a) from the second group detected 94% of the examined strains is interesting because PspA has been reported by others to be a serological highly variable protein.