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Currently available pneumococcal vaccines were examined for contamination by pneumococcal surface protein A (PspA) and pneumococcal surface adhesin A (PsaA). PspA could be detected in some (but not all) lots of 23-valent polysaccharide vaccine. Many lots of pneumococcal vaccines, including the heptavalent conjugate vaccine, were found to elicit small (but variable) antibody responses to PspA, PsaA, or both. The degree of contamination was highly variable, and this should be considered in pneumococcal vaccine evaluations or when capsular polysaccharide is used for pneumococcal antibody assays. [ABSTRACT FROM AUTHOR]

Pneumococcal surface protein A (PspA) is the only pneumococcal surface protein known to strongly bind lactoferrin on the bacterial surface. In the absence of PspA Streptococcus pneumoniae becomes more susceptible to killing by human apolactoferrin (apo-hLf), the iron-free form of lactoferrin. In the present study we examined diverse strains of S. pneumoniae that differed by 2 logs in their susceptibility to apo-hLf. Among these strains, the amount of apo-hLf that bound to cell surface PspA correlated directly with the resistance of the strain to killing by apo-hLf. Moreover examination of different pspA alleles on shared genetic backgrounds revealed that those PspAs that bound more lactoferrin conferred greater resistance to killing by apo-hLf. The effects of capsule on killing of pneumococci by apo-hLf were generally small, but on one genetic background, however, the lack of capsule was associated with 4-times as much apo-hLf binding and 30-times more resistance to killing by apo-hLf. Overall these finding strongly support the hypothesis that most of the variation in the ability of apo-hLf is dependent on the variation in the binding of apo-hLf to surface PspA and this binding is dependent on variation in PspA as well as variation in capsule which may enhance killing by reducing the binding of apo-hLf to PspA., (Published by Elsevier Ltd.)

Immunization with the pneumococcal proteins pneumolysin (Ply), choline binding protein A (CbpA), or pneumococcal surface protein A (PspA) elicits protective responses against invasive pneumococcal disease in animal models. In this study, we used different mouse models to test the efficacy of a variety of multivalent protein-based vaccines that comprised various combinations of full-length or peptide regions of the immunogens Ply, CbpA, or PspA: Ply toxoid with the L460D substitution (referred to herein as L460D); L460D fused with protective peptide epitopes from CbpA (YPT-L460D-NEEK [YLN]); L460D fused with the CD2 peptide containing the proline-rich region (PRR) of PspA (CD2-L460D); a combination of L460D and H70 (L460D+H70), a slightly larger PspA-derived peptide containing the PRR and the SM1 region; H70+YLN; and other combinations. Each mouse was immunized either intraperitoneally (i.p.) or subcutaneously (s.c.) with three doses (at 2-week intervals) of the various antigen combinations in alum adjuvant and then challenged in mouse models featuring different infection routes with multiple Streptococcus pneumoniae strains. In the i.p. infection sepsis model, H70+YLN consistently provided significant protection against three different challenge strains (serotypes 1, 2, and 6A); the CD2+YLN and H70+L460D combinations also elicited significant protection. Protection against intravenous (i.v.) sepsis (type 3 and 6A challenge strains) was largely dependent on PspA-derived antigen components, and the most protection was elicited by H70 with or without L460D or YLN. In a type 4 intratracheal (i.t.) challenge model that results in progression to meningitis, antigen combinations that contained YLN elicited the strongest protection. Thus, the trivalent antigen combination of H70+YLN elicited the strongest and broadest protection in diverse pneumococcal challenge models., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Pneumococcal surface adhesin A (PsaA) is a multifunctional lipoprotein known to bind nasopharyngeal epithelial cells, and is significantly involved in bacterial adherence and virulence. Identification of PsaA peptides that optimally bind human leucocyte antigen (HLA) and elicit a potent immune response would be of great importance to vaccine development. However, this is hindered by the multitude of HLA polymorphisms in humans. To identify the conserved immunodominant epitopes, we used an experimental dataset of 28 PsaA synthetic peptides and in silico methods to predict specific peptide-binding to HLA and murine MHC class II molecules. We also characterized spleen and cervical lymph node (CLN) -derived T helper (Th) lymphocyte cytokine responses to these peptides after Streptococcus pneumoniae strain EF3030 challenge in mice. Individual, yet overlapping, peptides 15 amino acids in length revealed residues of PsaA that consistently caused the highest interferon-γ, interleukin-2 (IL-2), IL-5 and IL-17 responses and proliferation as well as moderate IL-10 and IL-4 responses by ex vivo re-stimulated splenic and CLN CD4⁺ T cells isolated from S. pneumoniae strain EF3030-challenged F1 (B6 × BALB/c) mice. In silico analysis revealed that peptides from PsaA may interact with a broad range of HLA-DP, -DQ and -DR alleles, due in part to regions lacking β-turns and asparagine endopeptidase sites. These data suggest that Th cell peptides (7, 19, 20, 22, 23 and 24) screened for secondary structures and MHC class II peptide-binding affinities can elicit T helper cytokine and proliferative responses to PsaA peptides., (© 2013 John Wiley & Sons Ltd.)

Background: Streptococcus pneumoniae is a leading cause of childhood morbidity and mortality worldwide, despite the availability of effective pneumococcal vaccines. Understanding the molecular interactions between the bacterium and the host will contribute to the control and prevention of pneumococcal disease., Results: We used a combination of adherence assays, mutagenesis and functional genomics to identify novel factors involved in adherence. By contrasting these processes in two pneumococcal strains, TIGR4 and G54, we showed that adherence and invasion capacities vary markedly by strain. Electron microscopy showed more adherent bacteria in association with membranous pseudopodia in the TIGR4 strain. Operons for cell wall phosphorylcholine incorporation (lic), manganese transport (psa) and phosphate utilization (phn) were up-regulated in both strains on exposure to epithelial cells. Pneumolysin, pili, stress protection genes (adhC-czcD) and genes of the type II fatty acid synthesis pathway were highly expressed in the naturally more invasive strain, TIGR4. Deletion mutagenesis of five gene regions identified as regulated in this study revealed attenuation in adherence. Most strikingly, ∆SP_1922 which was predicted to contain a B-cell epitope and revealed significant attenuation in adherence, appeared to be expressed as a part of an operon that includes the gene encoding the cytoplasmic pore-forming toxin and vaccine candidate, pneumolysin., Conclusion: This work identifies a list of novel potential pneumococcal adherence determinants.

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.

Pneumococcal surface protein A (PspA) is a surface molecule on pneumococci that is required for full virulence in mouse models of infection. PspA has been reported to inhibit complement deposition on the pneumococcal surface. It has been assumed that this decreased complement deposition results in the inefficient phagocytosis of wild-type pneumococci. However, an effect of PspA on phagocytosis had not been shown. Our present studies demonstrated that a loss of PspA by capsular type 3 strains WU2 and A66.1 led to enhanced complement-dependent phagocytosis of the pneumococci by the mouse macrophage cell line J774A.1. This observation was made using human complement as well as mouse complement. Since this enhanced phagocytosis could be blocked by antibody to complement receptor CR3 on J774A.1, it was concluded that PspA's effect on phagocytosis was due to its effect on the amount of deposited complement, which in turn helped opsonize the pneumococci for phagocytosis. Since these studies included new independent mutants lacking PspA, the results provide solid confirmation of the previously reported effects of PspA on pneumococcal virulence and complement deposition. Finally, we showed that antibody to PspA, which is also known to enhance complement deposition, also enhances the phagocytosis of pneumococci in a largely complement-dependent manner.

Targeting an antigen to Fc receptors (FcR) can enhance the immune response to the antigen in the absence of adjuvant. Furthermore, we recently demonstrated that intranasal immunization with an FcγR-targeted antigen enhances protection against a category A intracellular mucosal pathogen, Francisella tularensis. To determine if a similar strategy could be applied to the important pathogen Streptococcus pneumoniae, we used an improved mucosal FcR-targeting strategy that specifically targets human FcγR type I (hFcγRI). A humanized single-chain antibody component in which the variable domain binds to hFcγRI [anti-hFcγRI (H22)] was linked in a fusion protein with the pneumococcal surface protein A (PspA). PspA is known to elicit protection against pneumococcal sepsis, carriage, and pneumonia in mouse models when administered with adjuvants. Anti-hFcγRI-PspA or recombinant PspA (rPspA) alone was used to intranasally immunize wild-type (WT) and hFcγRI transgenic (Tg) mice in the absence of adjuvant. The hFcγRI Tg mice receiving anti-hFcγRI-PspA exhibited elevated S. pneumoniae-specific IgA, IgG2c, and IgG1 antibodies in serum and bronchoalveolar lavage fluid. Neither immunogen was effective in protecting WT mice in the absence of adjuvant, but when PspA was targeted to hFcγRI as the anti-hFcγRI-PspA fusion, enhanced protection against lethal S. pneumoniae challenge was observed in the hFcγRI Tg mice compared to mice given nontargeted rPspA alone. Immune sera from the anti-hFcγRI-PspA-immunized Tg mice showed enhanced complement C3 deposition on bacterial surfaces, and protection was dependent upon an active complement system. Immune serum also showed an enhanced bactericidal activity directed against S. pneumoniae that appears to be lactoferrin mediated.

Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Several antigens, in addition to the polysaccharide capsule, have been implicated in both the virulence and protective immunity against Streptococcus pneumoniae; one of the best-studied S. pneumoniae antigens is pneumococcal surface protein A (PspA). Recently, it was shown that genetic polymorphisms could diminish CCL5 expression, which results in increased susceptibility to and progression of infectious diseases. We previously showed CCL5 blockade reduced PspA-specific humoral and cellular pneumococcal immunity, during S. pneumoniae strain EF3030-induced carriage, by diminishing IFN-γ and enhancing IL-10 secretion by effector T cells. We also identified immuno-dominant helper T lymphocyte (HTL) epitopes in PspA peptide 19-23 (PspA(199-246)), which caused comparatively more cytokine secretion and proliferation responses by splenic and cervical lymph node (CLN) CD4(+) T cells from mice previously challenged with S. pneumoniae strain EF3030. In this study, we sought to determine if PspA(199-246)-specific CD4(+) T cells responses were resistant to the effect of CCL5 deficiency. In short, T cell responses against these HTL epitopes were resistant to CCL5 inhibition, than compared to cells from control or naïve mice, and unaffected by reduced co-stimulatory molecule expression caused by CCL5 blockade. CCL5 deficiency also corresponded with a higher number of IL-10(+) CD11b(+) CD11c(Lo) and CD11b(+) CD11c(Hi) cells and lower IFN-γ expression by similar cells, than compared to controls. These data confirm CCL5 is an essential factor for optimal pneumococcal adaptive immunity and show CD4(+) T cell responses to PspA(199-246) are largely resistant to CCL5 deficiency., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Streptococcus pneumoniae is a significant pathogen capable of expressing protective and antigenically diverse capsules. To better understand the molecular basis of capsular antigenic diversity, we investigated the hypothetical serological role of wcjE, which encodes a capsule O-acetyltransferase, in the vaccine-targeted serotype 9V and related serotype 9A., Methods: We inactivated wcjE by recombination in a serotype 9V strain and determined wcjE sequences of 11 serotype 9A clinical isolates. We determined the antigenic phenotypes of these pneumococcal strains with serogroup 9-specific antibodies and flow cytometry., Results: Inactivation of wcjE in a serotype 9V strain resulted in expression of the 9A phenotype. Each serotype 9A clinical isolate contained a distinct mutation to wcjE. Flow cytometry showed that some 9A isolates (herein named 9Aα) expressed trace amounts of 9V-specific epitopes whereas others (named 9Aβ) did not express any. Recombination with 9Aα wcjE alleles into a 9Aβ strain conferred partial expression of 9V-specific epitopes., Conclusions: Each serotype 9A strain independently arose from a serotype 9V strain. Furthermore, clinical isolates identified as 9A can contain mutations to wcjE that are either partially functional or completely nonfunctional, demonstrating a previously unidentified antigenic heterogeneity of serotype 9A isolates.

We have previously shown that a pneumococcal surface protein A (PspA)-based vaccine containing DNA plasmid encoding the Flt3 ligand (FL) gene (pFL) as a nasal adjuvant prevented nasal carriage of Streptococcus pneumoniae. In this study, we further investigated the safety and efficacy of this nasal vaccine for the induction of PspA-specific antibody (Ab) responses against lung infection with S. pneumoniae. C57BL/6 mice were nasally immunized with recombinant PspA/Rx1 (rPspA) plus pFL three times at weekly intervals. When dynamic translocation of pFL was initially examined, nasal pFL was taken up by nasal dendritic cells (DCs) and epithelial cells (nECs) but not in the central nervous systems, including olfactory nerve and epithelium. Of importance, nasal pFL induced FL protein synthesis with minimum levels of inflammatory cytokines in the nasal washes (NWs) and bronchoalveolar lavage fluid (BALF). NWs and BALF as well as plasma of mice given nasal rPspA plus pFL contained increased levels of rPspA-specific secretory IgA and IgG Ab responses that were correlated with elevated numbers of CD8(+) and CD11b(+) DCs and interleukin 2 (IL-2)- and IL-4-producing CD4(+) T cells in the nasal mucosa-associated lymphoid tissues (NALT) and cervical lymph nodes (CLNs). The in vivo protection by rPspA-specific Abs was evident in markedly reduced numbers of CFU in the lungs, airway secretions, and blood when mice were nasally challenged with Streptococcus pneumoniae WU2. Our findings show that nasal pFL is a safe and effective mucosal adjuvant for the enhancement of bacterial antigen (Ag) (rPspA)-specific protective immunity through DC-induced Th2-type and IL-2 cytokine responses.

It is known that apolactoferrin, the iron-free form of human lactoferrin, can kill many species of bacteria, including Streptococcus pneumoniae. Lactoferricin, an N-terminal peptide of apolactoferrin, and fragments of it are even more bactericidal than apolactoferrin. In this study we found that apolactoferrin must be cleaved by a serine protease in order for it to kill pneumococci. The serine protease inhibitors were able to block killing by apolactoferrin but did not block killing by a lactoferrin-derived peptide. Thus, the killing of pneumococci by apolactoferrin appears to require a protease to release a lactoferricin-like peptide(s). Incubation of apolactoferrin with growing pneumococci resulted in a 12-kDa reduction in its molecular mass, of which about 7 to 8 kDa of the reduction was protease dependent. Capsular type 2 and 19F strains with mutations in the gene encoding the major cell wall-associated serine protease, prtA, lost much of their ability to degrade apolactoferrin and were relatively resistant to killing by apolactoferrin (P < 0.001). Recombinant PrtA was also able to cleave apolactoferrin, reducing its mass by about 8 kDa, and greatly enhance the killing activity of the solution containing the apolactoferrin and its cleavage products. Mass spectroscopy revealed that PrtA makes a major cut between amino acids 78 and 79 of human lactoferrin, removing the N-terminal end of the molecule (about 8.6 kDa). The simplest interpretation of these data is that the mechanism by which apolactoferrin kills Streptococcus pneumoniae requires the release of a lactoferricin-like peptide(s) and that it is this peptide(s), and not the intact apolactoferrin, which kills pneumococci.

Pneumococcal phase variation of 37 middle ear and 31 nasopharyngeal isolates obtained from children with acute otitis media was examined in the absence of intervening culture. The fraction of the opaque colonies was significantly higher in middle ear isolates than in nasopharyngeal isolates. The difference is probably the result of the pneumococci adapting to differential selective environments.

Our previous study showed that a combination of a plasmid-expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotides (CpG ODN) as a combined nasal adjuvant elicited mucosal immune responses in aged (2-y-old) mice. In this study, we investigated whether a combination of pFL and CpG ODN as a nasal adjuvant for a pneumococcal surface protein A (PspA) would enhance PspA-specific secretory-IgA Ab responses, which could provide protective mucosal immunity against Streptococcus pneumoniae infection in aged mice. Nasal immunization with PspA plus a combination of pFL and CpG ODN elicited elevated levels of PspA-specific secretory-IgA Ab responses in external secretions and plasma in both young adult and aged mice. Significant levels of PspA-specific CD4(+) T cell proliferative and PspA-induced Th1- and Th2- type cytokine responses were noted in nasopharyngeal-associated lymphoreticular tissue, cervical lymph nodes, and spleen of aged mice, which were equivalent to those in young adult mice. Additionally, increased numbers of mature-type CD8, CD11b-expressing dendritic cells were detected in mucosal inductive and effector lymphoid tissues of aged mice. Importantly, aged mice given PspA plus a combination of pFL and CpG ODN showed protective immunity against nasal S. pneumoniae colonization. These results demonstrate that nasal delivery of a combined DNA adjuvant offers an attractive possibility for protection against S. pneumoniae in the elderly.

Streptococcus pneumoniae expressing serogroup 6 capsules frequently causes pneumococcal infections and the evolutionary origins of the serogroup 6 strains have been extensively studied. However, these studies were performed when serogroup 6 had only two known members (serotypes 6A and 6B) and before the two new members (serotypes 6C and 6D) expressing wciN(β) were found. We have therefore reinvestigated the evolutionary origins of serogroup 6 by examining the profiles of the capsule gene loci and the multilocus sequence types (MLSTs) of many serogroup 6 isolates from several continents. We confirmed that there are two classes of cps locus sequences for serogroup 6 isolates. In our study, class 2 cps sequences were limited to a few serotype 6B isolates. Neighbour-joining analysis of cps sequence profiles showed a distinct clade for 6C and moderately distinct clades for class 1 6A and 6B sequences. The serotype 6D cps profile was found within the class 1 6B clade, suggesting that it was created by recombination between 6C and 6B cps loci. Interestingly, all 6C isolates also had a unique wzy allele with a 6 bp deletion. This suggests that serotype switching to 6C involves the transfer of a large (>4 kb) gene segment that includes both the wciN(β) allele and the 'short' wzy allele. The MLST studies of serotype 6C isolates suggest that the 6C cps locus is incorporated into many different pneumococcal genomic backgrounds but that, interestingly, 6C cps may have preferentially entered strains of the same genomic backgrounds as those of serotype 6A.

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.

Streptococcus pneumoniae is one of the important causative pathogens for both upper and lower respiratory tract infections during childhood. The current study was designed to evaluate the protection against fatal pneumococcal infections during the infant period by maternal immunization with pneumococcal surface protein A (PspA). Four-week-old females BALB/c mice were immunized with PspA and cholera toxin B (CTB) intranasally twice a week for 3 weeks. After mating, the 10-day-old offspring of these mice were intraperitoneally (i.p.) infected with S. pneumoniae to evaluate survival. Anti-PspA-specific IgG antibody was induced in the sera of mother and offspring. The survival times to death after systemic fatal pneumococcal infections were significantly extended among offspring delivered from PspA-immunized mothers than the controls. Current findings suggest that maternal intranasal immunization with PspA is an attractive procedure against pneumococcal infections in early childhood., (Copyright © 2011 S. Karger AG, Basel.)

IL-1α and IL-1β were evaluated for their ability to provide adjuvant activity for the induction of serum antibody responses when nasally administered with protein antigens in mice and rabbits. In mice, intranasal (i.n.) immunization with pneumococcal surface protein A (PspA) or tetanus toxoid (TT) combined with IL-1β induced protective immunity that was equivalent to that induced by parenteral immunization. Nasal immunization of awake (i.e., not anesthetized) rabbits with IL-1-adjuvanted vaccines induced highly variable serum antibody responses and was not as effective as parenteral immunization for the induction of antigen-specific serum IgG. However, i.n. immunization of deeply anesthetized rabbits with rPA+IL-1α consistently induced rPA-specific serum IgG ELISA titers that were not significantly different than those induced by intramuscular (IM) immunization with rPA+alum although lethal toxin-neutralizing titers induced by nasal immunization were lower than those induced by IM immunization. Gamma scintigraphy demonstrated that the enhanced immunogenicity of nasal immunization in anesthetized rabbits correlated with an increased nasal retention of i.n. delivered non-permeable radio-labeled colloidal particles. Our results demonstrate that, in mice, IL-1 is an effective adjuvant for nasally administered vaccines for the induction of protective systemic immunity and that in non-rodent species, effective induction of systemic immunity with nasally administered vaccines may require formulations that ensure adequate retention of the vaccine within the nasal cavity., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

This study was designed to investigate whether secretory-IgA (S-IgA) Abs induced by a pneumococcal surface protein A (PspA)-based nasal vaccine are necessary for prevention of streptococcal colonization. Mice nasally immunized with PspA plus a plasmid expressing Flt3 ligand (pFL) cDNA as a mucosal adjuvant showed significantly higher levels of PspA-specific S-IgA and IgG Ab responses in both plasma and nasal washes when compared with naive mice. Although IgA(-/-) mice given nasal PspA plus pFL had significantly high levels of PspA-specific IgG Abs, high numbers of CFUs were detected in nasal washes and nasal passages. In contrast, vaccinated wild-type mice showed essentially no bacteria in the nasal cavity. Further, a nasal vaccine consisting of PspA plus pFL effectively reduced pre-existing Streptococcus pneumoniae in the nasal cavity. These results show that PspA-based vaccine-induced specific S-IgA Abs play a necessary role in the regulation of S. pneumoniae colonization in the nasal cavity.

Pneumococcal surface protein A (PspA) and PspC of Streptococcus pneumoniae are surface virulence proteins that interfere with complement deposition and elicit protective immune responses. The C-terminal halves of PspA and PspC have some structural similarity and contain highly cross-reactive proline-rich (PR) regions. In many PR regions of PspA and PspC, there exists an almost invariant nonproline block (NPB) of about 33 amino acids. Neither the PR regions nor their NPB exhibit the alpha-helical structure characteristic of much of the protection-eliciting N-terminal portions of PspA and PspC. Prior studies of PspA and PspC as immunogens focused primarily on the alpha-helical regions of these molecules that lack the PR and NPB regions. This report shows that immunization with recombinant PR (rPR) molecules and passive immunization with monoclonal antibodies reactive with either NPB or PR epitopes are protective against infection in mice. PR regions of both PspA and PspC were antibody accessible on the pneumococcal surface. Our results indicate that while PspA could serve as a target of these protective antibodies in invasive infections, PspC might not. When antibody responses to rPR immunogens were evaluated by using flow cytometry to measure antibody binding to live pneumococci, it was observed that the mice that survived subsequent challenge produced significantly higher levels of antibodies reactive with exposed PR epitopes than the mice that became moribund. Due to their conservation and cross-reactivity, the PR regions and NPB regions represent potential vaccine targets capable of eliciting cross-protection immunity against pneumococcal infection.

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-gamma, 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 F(1) (B6xBALB/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.

Low yields, poor folding efficiencies and improper disulfide bridge formation limit large-scale production of cysteine-rich proteins in Escherichia coli. Human renal dipeptidase (MDP), the only human beta-lactamase known to date, is a homodimeric enzyme, which contains six cysteine residues per monomer. It hydrolyses penem and carbapenem beta-lactam antibiotics and can cleave dipeptides containing amino acids in both D: - and L: -configurations. In this study, MDP accumulated in inactive form in high molecular weight, disulfide-linked aggregates when produced in the E. coli periplasm. Mutagenesis of Cys361 that mediates dimer formation and Cys93 that is unpaired in the native MDP led to production of soluble recombinant enzyme, with no change in activity compared with the wild-type enzyme. The removal of unpaired or structurally inessential cysteine residues in this manner may allow functional production of many multiply disulfide-linked recombinant proteins in E. coli.

Streptococcus pneumoniae is a major bacterial respiratory pathogen. Current licensed pneumococcal polysaccharide and polysaccharide-protein conjugate vaccines are administered by an intramuscular injection. In order to develop a new-generation vaccine that can be administered in a needle-free mucosal manner, we have constructed early 1 and 3 gene regions (E1/E3) deleted, replication-defective adenoviral vectors encoding pneumococcal surface antigen A (PsaA), the N-fragment of pneumococcal surface protein A (N-PspA), and the detoxified mutant pneumolysin (PdB) from S. pneumoniae strain D39. Intranasal vaccination with the three adenoviral vectors (Ad/PsaA, Ad/N-PspA, and Ad/PdB) in mice resulted in robust antigen-specific serum immunoglobulin G responses, as demonstrated by an enzyme-linked immunosorbent assay. In addition, nasal mucosal vaccination with the combination of the three adenoviral vectors conferred protection against S. pneumoniae strain D39 colonization in mouse lungs. Taken together, these data demonstrate the feasibility of developing a mucosal vaccine against S. pneumoniae using recombinant adenoviruses for antigen delivery.

Background: Streptococcus pneumoniae (pneumococcus) causes respiratory and systemic infections that are a major public health problem worldwide. It has been postulated that pneumococci persist in vivo in biofilm communities., Methods: In this study, we analyzed whether pneumococci form biofilms in vivo, and if so, whether biofilms correlated with bacterial persistence. Chinchillas were infected with S. pneumoniae TIGR4 and euthanized at varying times after infection, after which the superior ear bullae were excised and examined by culture and microscopy., Results: Dense material, resembling the biofilms of other otitis media pathogens, was visible in the middle ear as late as 12 days after infection. Scanning electron microscopy revealed bacteria within an electron-dense matrix, similar to pneumococcal biofilms formed in vitro. Viability staining revealed groups of viable diplococci, as well as viable and nonviable host cells, attached to a fibrous matrix that was positive when stained with propidium iodide. Cryosections of biofilms were treated with polyclonal antibodies against the pneumococcal surface components pneumococcal surface protein A family 2, pneumococcal surface protein C, choline-binding protein, and neuraminidase, coupled with appropriate secondary antibody conjugates. Immunofluorescent staining showed the presence of pneumococcal communities within the material recovered from the middle ear chamber., Conclusions: On the basis of these data, we conclude that pneumococci form biofilms in vivo and that this process may be intertwined with the formation of neutrophil extracellular traps. These findings provide new insights into the potential causes of antibiotic treatment failure and bacterial persistence in chronic pneumococcal otitis media.

Following introduction of the 7-valent pneumococcal conjugate vaccine to the United States, rates of invasive pneumococcal disease (IPD) caused by serotype 6A declined among all age groups, while rates of IPD caused by newly identified serotype 6C increased slightly among persons 5 years of age and older. Conventionally serotyped 6A isolates (CS6As) from active population-based surveillance during 1999 and 2006 to 2007 were classified as serotypes 6A and 6C by an expedient and highly accurate serotype 6C-specific PCR assay developed during this study. PCR testing of 636 year 1999, 2006, and 2007 CS6As revealed 6C proportions of 35/214 (16.4%), 141/218 (64.7%), and 141/204 (69.1%), respectively. These results agreed with those from a previously devised monoclonal antibody-based serotyping system (346 CS6As compared). Type 6C IPD incidence significantly increased during 2006 and 2007 compared to during 1999 (0.57 to 0.58 cases per 100,000 and 0.22 cases per 100,000, respectively; 164% increase from 1999 to 2007 [95% confidence interval, 87 to 270%]), while rates of IPD due to types 6A and 6B markedly decreased. In 2007, 31.2% of 6C isolates were not susceptible to penicillin. Serotype 6C is now the predominant serotype associated with serogroup 6 IPD in the United States and is often penicillin nonsusceptible. We performed multilocus sequence typing (MLST) on a limited sampling of 6C isolates with different antimicrobial susceptibility profiles. MLST of 42 6C isolates revealed 12 genotypes distributed among six distinct genetic groups. Fifteen 6C isolates shared one of four different MLST types with 6C-negative CS6As. MLST results suggest 6C strains arose from independent recombination events involving only serotype 6A and 6C parental strains.

Serogroup 6 of Streptococcus pneumoniae contains three serotypes, named 6A, 6B and 6C, with highly homologous capsule gene loci. The 6A and 6B capsule gene loci consistently differ from each other by only one nucleotide in the wciP gene. The 6A capsule gene locus has a galactosyltransferase, which has been replaced with a glucosyltransferase in the 6C capsule gene locus. We considered that a new serotype named '6X1' would be possible if the galactosyltransferase of the 6B capsule gene locus is replaced with the glucosyltransferase of 6C. We demonstrate that this gene transfer yields a viable pneumococcal strain and that the capsular polysaccharide (PS) from this strain has the predicted chemical structure and serological similarity to the capsular PS of the 6B serotype. The new strain (i.e. serotype 6X1) is typed as 6B by the quellung reaction, but it can be distinguished from 6B strains with mAbs to 6B PS. Reexamination of 264 pneumococcal isolates that had been previously typed as 6B with classical typing methods revealed no isolates expressing serotype 6X1. Nevertheless, this study shows that this capsular PS is biochemically possible and could exist/emerge in nature.

Streptococcus pneumoniae has been shown to bind to erythrocytes via a process called immune adherence. This adherence and the subsequent transfer of pneumococci from erythrocytes to macrophages are both dependent on complement C3 deposition onto the pneumococcal surface. The observation that anti-capsule antibody increases C3 deposition on the pneumococcal capsule indicated that anti-capsule antibody may also facilitate the clearance of pneumococci through immune adherence. Using pneumococcal strain WU2 (capsule type 3) and its nonencapsulated mutant JD908, we found that monoclonal antibody (MAb) to type 3 capsule increases complement C3, C1q, and C4 deposition on WU2 and enhanced the immune adherence of WU2 to erythrocytes. The MAb to type 3 capsule also enhanced the transfer of WU2 from erythrocytes to macrophages. Moreover, the transfer reaction was inhibited by preincubating macrophages with anti-CR3 or anti-Fc gammaRIII/II MAb, indicating that CR3 and Fc gammaRIII/II on macrophages mediate this process. The transfer reactions of JD908 (opsonized with complement) and WU2 (opsonized with complement plus MAb to type 3 capsule) were similarly inhibited by anti-CR3 MAb, but only the latter was inhibited by anti-Fc gammaRIII/II MAb. This finding indicates that although complement and the macrophage receptor CR3 are essential for the transfer reaction, if antibody is present it can further enhance the transfer reaction through a process dependent on Fc gammaRIII/II. Using pre- and postvaccination sera of people immunized with the 23-valent pneumococcal polysaccharide vaccine, we confirmed that human anti-capsule antibodies are also able to increase the immune adherence of pneumococci and their transfer to macrophages.

PspA is a structurally variable surface protein important to the virulence of pneumococci. PspAs are serologically cross-reactive and exist as two major families. In this study, we determined the distribution of PspA families 1 and 2 among pneumococcal strains isolated from the middle ear fluid (MEF) of children with acute otitis media and from nasopharyngeal specimens of children with pneumococcal carriage. We characterized the association between the two PspA families, capsular serotypes, and multilocus sequence types (STs) of the pneumococcal isolates. MEF isolates (n = 201) of 109 patients and nasopharyngeal isolates (n = 173) of 49 children were PspA family typed by whole-cell enzyme immunoassay (EIA). Genetic typing (PCR) of PspA family was done for 60 isolates to confirm EIA typing results. The prevalences of PspA families 1 and 2 were similar among pneumococci isolated from MEF (51% and 45%, respectively) and nasopharyngeal specimens (48% each). Isolates of certain capsule types as well as isolates of certain STs showed statistical associations with either family 1 or family 2 PspA. Pneumococci from seven children with multiple pneumococcal isolates appeared to express serologically different PspA families in different isolates of the same serotype; in three of the children the STs of the isolates were the same, suggesting that antigenic changes in the PspA expressed may have taken place. The majority of the isolates (97%) belonged to either PspA family 1 or family 2, suggesting that a combination including the two main PspA families would make a good vaccine candidate.

Pneumococcal surface protein A (PspA) is an important virulence factor of Streptococcus pneumoniae. PspA exists as two major families, which include variable but serologically cross-reactive proteins. Previous studies with a family 1 PspA antigen suggested that children develop low concentrations of anti-PspA after pneumococcal carriage or infection. In this study, antibody to PspA families 1 and 2 was measured by an enzyme immunoassay of the serum and saliva of children with a history of culture-proven pneumococcal colonization and/or acute otitis media and in the serum and saliva of adults. The PspA families of the pneumococcal strains isolated from children were determined. The majority of the children had high serum and salivary anti-PspA concentrations to the PspA family they had encountered and low concentrations to the other, whereas adults had high antibody concentrations to both PspA families, both in serum and in saliva. The results suggest that children have a relatively family-specific antibody response to the PspA family they have been exposed to and that any PspA vaccine for children should contain members of both major PspA families.

The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that often exhibit distinct structural changes that modify their pore-forming activity. A soluble platelet aggregation factor from Streptococcus mitis (Sm-hPAF) was characterized and shown to be a functional CDC with an amino-terminal fucose-binding lectin domain. Sm-hPAF, or lectinolysin (LLY) as renamed herein, is most closely related to CDCs from Streptococcus intermedius (ILY) and Streptococcus pneumoniae (pneumolysin or PLY). The LLY gene was identified in strains of S. mitis, S. pneumoniae, and Streptococcus pseudopneumoniae. LLY induces pore-dependent changes in the light scattering properties of the platelets that mimic those induced by platelet aggregation but does not induce platelet aggregation. LLY monomers form the typical large homooligomeric membrane pore complex observed for the CDCs. The pore-forming activity of LLY on platelets is modulated by the amino-terminal lectin domain, a structure that is not present in other CDCs. Glycan microarray analysis showed the lectin domain is specific for difucosylated glycans within Lewis b (Le (b)) and Lewis y (Le (y)) antigens. The glycan-binding site is occluded in the soluble monomer of LLY but is apparently exposed after cell binding, since it significantly increases LLY pore-forming activity in a glycan-dependent manner. Hence, LLY represents a new class of CDC whose pore-forming mechanism is modulated by a glycan-binding domain.

Previous studies have suggested that pneumococcal choline binding protein A (PcpA) is important for the full virulence of Streptococcus pneumoniae, and its amino acid sequence suggests that it may play a role in cellular adherence. PcpA is under the control of a manganese-dependent regulator and is only expressed at low manganese concentrations, similar to those found in the blood and lungs. PcpA expression is repressed under high manganese concentrations, similar to those found in secretions. In this study, we have demonstrated that PcpA elicits statistically significant protection in murine models of pneumonia and sepsis. In the model of pneumonia with each of four challenge strains, statistically fewer S. pneumoniae cells were recovered from the lungs of mice immunized with PcpA and alum versus mice immunized with alum only. The immunizations reduced the median CFU by 4- to 400-fold (average of 28-fold). In the model of sepsis using strain TIGR4, PcpA expression resulted in shorter times to become moribund and subcutaneous immunization with PcpA increased survival times of mice infected with wild-type PcpA-expressing pneumococci.

Pneumococcal surface protein A (PspA) and PspC are important virulence factors. Their absence has been shown to allow improved clearance of pneumococci from the blood of mice and to decrease pneumococcal virulence. In the presence of antibody and complement, pneumococci attach to erythrocytes in a process called immune adherence (IA), which facilitates their delivery to, and eventual phagocytosis by, macrophages. It is not known, however, if PspA and PspC affect IA. Using PspA and/or PspC isogenic mutants and complement-deficient mouse sera, we demonstrated that absence of PspA allows greater deposition of C1q and thus increased classical-pathway-mediated C3 deposition. In the absence of both PspA and PspC, there is also a major increase in C1q-independent C3 deposition through the alternative pathway. The latter was observed even though absence of PspC alone did not have a major effect on alternative-pathway-dependent complement deposition. The enhanced complement C3 deposition realized in the absence of PspA alone and in the absence of PspA and PspC resulted in both greatly increased IA to human erythrocytes and improved transfer of pneumococci from erythrocytes to phagocytes. These data provide new insight into how PspA and PspC act in synergy to protect pneumococci from complement-dependent clearance during invasive infection.

PspA is an important pneumococcal vaccine candidate that is capable of inducing protection in different animal models. Because of its structural diversity, a PspA-based vaccine should contain at least one fragment from each of the two major families (1 and 2) in order to elicit broader protection. In the present work, we have tested the potential of PspA hybrids containing fused portions of family 1 and 2 (PspA1ABC-4B and PspA1ABC-3AB) PspA fragments to induce protection against pneumococci bearing distinct PspA fragments. Sera from mice immunized with these hybrid PspA fragments were able to increase C3 deposition on pneumococci bearing PspA fragments from both families, in contrast with sera made against the PspA family 1 (PspA1ABC) and PspA family 2 (PspA3ABC) fragments, which were effective only within the same family. Although PspA hybrids were able to extend protection against pneumococcal infection with strains bearing diverse PspA fragments, the immunity elicited by family 2 was clade dependent, suggesting that PspA fragments from family 2 clades 3 and 4 should both be included in a comprehensive PspA vaccine. These results indicate that PspA fusion proteins constitute an efficient immunization strategy for future PspA-based antipneumococcal vaccines since they are able to extend protection provided by a protein derived from a single transcript.

Background: Pneumococcal surface protein A (PspA) is a highly variable yet cross-reactive protein that exists as 2 major families. We assessed the development of human serum and salivary antibodies against the PspA families 1 (PspA1) and 2 (PspA2) in early childhood and their role in the prevention of pneumococcal acute otitis media (AOM)., Methods: Serum levels of IgG and salivary levels of IgA antibodies to PspA1 and PspA2 were measured by use of enzyme immunoassay from the samples from the Finnish Otitis Media Cohort Study obtained at the ages of 12 months (287 and 160 samples, respectively) and 18 months (258 and 131 samples, respectively). The Cox proportional hazard model was used to evaluate the relative risk (RR) of pneumococcal AOM during the 6 months after sampling relative to concentration of serum or presence of salivary anti-PspA in the samples., Results: Anti-PspA1 and anti-PspA2 concentrations at 12 and 18 months were related to prior culture-confirmed pneumococcal exposure. The concentrations of serum anti-PspA were not significantly associated with the risk of pneumococcal AOM. At 18 months, the presence of salivary anti-PspA was significantly associated with a lower risk of pneumococcal AOM during the 6 months after sampling (RR, 0.27 [95% confidence interval, 0.11-0.69])., Conclusions: The lowered risk of pneumococcal AOM associated with the presence of salivary anti-PspA at 18 months suggests that mucosal anti-PspA antibodies have a role in the prevention of pneumococcal AOM.

Immunity to pneumococcal colonization in mice by exposure to live or killed pneumococci has been shown to be antibody independent but dependent on CD4+ T cells. Here we show that intranasal immunization with pneumococcal proteins (pneumococcal surface protein C, adhesin A, and a pneumolysoid) can elicit a similar mechanism of protection. Colonization could be significantly reduced in mice congenitally deficient in immunoglobulins after intranasal immunization with this mixture of proteins; conversely, the depletion of CD4+ T cells in immunized wild-type mice at the time of challenge eliminated the protection afforded by immunization. Overall, our results show that intranasal immunization with a mixture of pneumococcal proteins protects against colonization in an antibody-independent, CD4+ T-cell-dependent manner.

We have recently reported a new pneumococcal serotype (6C), which is closely related to serotype 6A (I. H. Park et al., J. Clin. Microbiol. 45:1225-1233, 2007). To investigate the genetic basis for serotype 6C, we studied the capsule gene loci of 14 6C isolates from three different continents, including one isolated in Alabama 27 years ago. The wciN region of all 6C isolates has a 1,029-bp-long sequence that replaces the 1,222-bp-long sequence of the 6A wciN region. This recombination event has created a new 1,125-bp-long open reading frame which encodes a product that is also homologous to glycosyl transferases. Flanking this introduced gene is 300 bp upstream and 100 bp downstream with only about 90% homology with 6A and which is identical in all 6C isolates. Transfer of the wciN region converts 6A to 6C. Determination of the DNA sequence of the entire capsule gene locus of one 6C isolate showed that the 6C capsule gene locus is almost identical (>98% homologous) to that of 6A except for the wciN region. These findings indicate that the 6C capsule type originated more than 27 years ago by a single recombination event in a 6A locus in which 6A wciN was replaced by a gene of unknown origin.

Human lactoferrin, a component of the innate immune system, kills a wide variety of microorganisms including the Gram positive bacteria Streptococcus pneumoniae. Pneumococcal surface protein A (PspA) efficiently inhibits this bactericidal action. The crystal structure of a complex of the lactoferrin-binding domain of PspA with the N-lobe of human lactoferrin reveals direct and specific interactions between the negatively charged surface of PspA helices and the highly cationic lactoferricin moiety of lactoferrin. Binding of PspA blocks surface accessibility of this bactericidal peptide preventing it from penetrating the bacterial membrane. Results of site-directed mutagenesis, in vitro protein binding assays and isothermal titration calorimetry measurements corroborate that the specific electrostatic interactions observed in the crystal structure represent major associations between PspA and lactoferrin. The structure provides a snapshot of the protective mechanism utilized by pathogens against the host's first line of defense. PspA represents a major virulence factor and a promising vaccine candidate. Insights from the structure of the complex have implications for designing therapeutic strategies for treatment and prevention of pneumococcal diseases that remain a major public health problem worldwide.

Several microbial pathogens stimulate the ectodomain shedding of host cell surface proteins to promote their pathogenesis. We reported previously that Pseudomonas aeruginosa and Staphylococcus aureus activate the ectodomain shedding of syndecan-1 and that syndecan-1 shedding promotes P. aeruginosa pathogenesis in mouse models of lung and burned skin infections. However, it remains to be determined whether activation of syndecan-1 shedding is a virulence mechanism broadly used by pathogens. Here we show that Streptococcus pneumoniae stimulates syndecan-1 shedding in cell culture-based assays. S. pneumoniae-induced syndecan-1 shedding was repressed by peptide hydroxamate inhibitors of metalloproteinases but not by inhibitors of intracellular signaling pathways previously found to be essential for syndecan-1 shedding caused by P. aeruginosa, S. aureus, or other shedding agonists. A 170-kDa protein fraction with a peptide hydroxamate-sensitive shedding activity was purified by ammonium sulfate precipitation, DEAE chromatography, and size exclusion chromatography. Mass spectrometry analyses revealed that the 170-kDa fraction is composed of ZmpB and ZmpC, two metalloproteinase virulence factors of S. pneumoniae. Both the purified 170-kDa ZmpB/ZmpC fraction and unfractionated S. pneumoniae culture supernatant generated syndecan-1 ectodomains that are smaller than those released by endogenous shedding. Further, a mutant S. pneumoniae strain deficient in zmpC, but not zmpB, lost its capacity to stimulate syndecan-1 shedding. These data demonstrate that S. pneumoniae directly sheds syndecan-1 ectodomains through the action of ZmpC.

One of the candidate proteins for a mucosal vaccine antigen against Streptococcus pneumoniae is PsaA (pneumococcal surface antigen A). Vaccines targeting mucosal immunity may raise concerns as to possible alterations in the normal microbiota, especially in the case of PsaA, which was shown to have homologs with elevated sequence identity in other viridans group streptococci. In this work, we demonstrate that intranasal immunization with a cholera toxin B subunit-PsaA fusion protein is able to protect mice against colonization with S. pneumoniae but does not significantly alter the natural oral or nasopharyngeal microbiota of mice.

Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Recently, it has been shown that genetic polymorphisms can result in diminished expression of CCL5, which results in increased susceptibility to and progression of infectious diseases. We show that CCL5, together with Th cytokine mRNA expression, is temporally up-regulated during pneumococcal carriage. To determine the contribution of CCL5 to pneumococcal surface antigen A-specific humoral and cellular pneumococcal immunity, mice were treated with anti-CCL5 or control Abs before and during Streptococcus pneumoniae strain EF3030-challenge for the initiation of carriage. CCL5 blockade resulted in a decrease of CD4(+) and CD8(+) T cells as well as CD11b(+) cells in the spleen, cervical lymph node, lung, and nasopharyngeal associated lymphoid tissue during the recognition phase of the pneumococcal adaptive immune response. CCL5 blockade significantly reduced the Ag-specific IgG2a and IgG1 Abs in serum and IgA Ab levels in nasal washes. These decreases also corresponded to reductions in Ag-specific T cell (mucosal and systemic) responses. CCL5 inhibition resulted in decreasing the quantity of IL-4- and IFN-gamma-secreting CD4(+) T cells and increasing the number of Ag-specific IL-10-producing CD4(+) T cells; these changes combined also corresponded with the transition from pneumococcal carriage to lethal pneumonia. These data suggest that CCL5 is an essential factor for the induction and maintenance of protective pneumococcal immunity.

The concentration of Mn2+ is 1,000-fold higher in secretions than it is at internal sites of the body, making it a potential signal by which bacteria can sense a shift from a mucosal environment to a more invasive site. PsaR, a metal-dependent regulator in Streptococcus pneumoniae, was found to negatively affect the transcription of psaBCA, pcpA, rrgA, rrgB, rrgC, srtBCD, and rlrA in the presence of Mn2+. psaBCA encode an ABC-type transporter for Mn2+. pcpA, rrgA, rrgB, and rrgC encode several outer surface proteins. srtBCD encode a cluster of sortase enzymes, and rlrA encodes a transcriptional regulator. Steady-state RNA levels are high under low Mn2+ concentrations in the wild-type strain and are elevated under both high and low Mn2+ concentrations in a psaR mutant strain. RlrA is an activator of rrgA, rrgB, rrgC, and srtBCD (D. Hava and A. Camilli, Mol. Microbiol. 45:1389-1406, 2002), suggesting that PsaR may indirectly control these genes through rlrA, while PsaR-dependent repression of psaBCA, pcpA, and rlrA transcription is direct. The impact of Mn2+-dependent regulation on virulence was further examined in mouse models of pneumonia and nasopharyngeal carriage. The abilities of DeltapsaR, pcpA, and DeltapsaR DeltapcpA mutant strains to colonize the lung were reduced compared to those of the wild type, confirming that both PcpA-mediated gene regulation and PsaR-mediated gene regulation are required for full virulence in the establishment of pneumonia. Neither PcpA nor PsaR was found to be required for colonization of the nasopharynx in a carriage model. This is the first demonstration of Mn2+ acting as a signal for the expression of virulence factors within different host sites.

We have recently developed a rapid pneumococcal serotyping method called "multibead assay" (J. Yu et al., J. Clin. Microbiol. 43:156-162, 2005) based on a multiplexed immunoassay for capsular polysaccharides in lysates of pneumococcal cultures. The multibead assay can identify 36 serotypes (1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/33C, 11A/11D/11F, 12A/12B/12F, 14, 15B/5C, 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F). More than 90% of the U.S. isolates express one of these serotypes (J. B. Robbins et al., J. Infect. Dis. 148:1136-1159, 1983). To validate the new assay, we examined 495 clinical isolates of pneumococci obtained in Brazil, Denmark, and Mexico. Pneumococci were serotyped by the Neufeld test in their countries of origin, and lysates of each strain were coded and mailed to the United States for the multibead assay at ambient temperature without any thermal protection. After breaking the code, 54 discrepancies (11% of samples) were noted, but 46 were due to nonreproducible technical problems or insufficient growth of the pneumococci. All of the isolates grew well for a second test, and therefore, the culture medium used for the multibead assay is adequate. The discrepancies persisted for eight isolates, involving the 6A, 11A, and 18C serotypes. Additional studies of the eight isolates showed that the discrepancies were due to differences in the reagents used in the multibead or Neufeld tests for these three serotypes. For instance, five isolates were typed as 6A with the Neufeld test but as nontypeable by the multibead assay. Selection of another new monoclonal antibody (Hyp6AG1) for the multibead assay resulted in all five discrepant isolates typing as 6A. This finding indicates the validity of the multibead assay and emphasizes the need to validate any new pneumococcal serotyping assay with a large number of clinical isolates from different locations. It also suggests the presence of serological subtypes among isolates expressing the 6A serotype.

The pneumococcal surface protein PspA, a cell-wall-associated surface protein, is a promising component for pneumococcal vaccines. In this study, the distribution of the PspA family was determined in a panel of invasive and clinically important pneumococcal isolates from adults over 50 years of age, collected between 1995 and 2002. One thousand eight hundred and forty-seven recent isolates from invasive pneumococcal disease were obtained from seven Western countries, together with clinical data. An ELISA-based serological method was standardized in order to determine the PspA family and clade distribution. Molecular tests were used when isolates were non-typable by ELISA (PspA family typing by PCR). Only 42 (2.3 %) isolates were non-typable by ELISA and PspA family typing by PCR was performed. Finally, 3 isolates were considered as non-pneumococcal and 1844 were classified as follows: 749 (40.6 %) were PspA family 1, 1078 (58.5 %) were PspA family 2, 13 (0.7 %) were PspA family 1 and 2 and 4 (0.2 %) remained non-typable. The cross-reactivity of antibodies to PspAs of different clades was confirmed. In conclusion, inclusion of PspA family 1 and family 2 in future pneumococcal vaccines would ensure broad coverage of pneumococcal strains infecting people over 50 years of age.

Asplenic individuals have increased susceptibility to septicemia caused by encapsulated bacteria. Streptococcus pneumoniae, a pathogen carried in the nasal passages of many humans without complication, is responsible for a large proportion of infections seen in asplenic individuals. Our studies have evaluated the efficacy of antibodies to pneumococcal surface protein A (PspA) in protection of asplenic mice. In passive immunity studies, pneumococci were more completely cleared from the blood of splenectomized mice receiving passive antiserum to PspA than those receiving normal rabbit serum. From active mucosal (intranasal) and systemic (subcutaneous) immunizations with rPspA, we determined that the levels of PspA antibodies produced in splenectomized mice were not significantly different from levels seen in mock-splenectomized animals. This active immunity to PspA was able to protect splenectomized mice against death following infection with live pneumococci. Our results suggest that PspA immunization may also protect asplenic humans from pneumococcal infections.

Fluoroquinolone resistance in Streptococcus pneumoniae is primarily mediated by point mutations in the quinolone resistance-determining regions of gyrA and parC. Antimicrobial resistance mutations in housekeeping genes often decrease fitness of microorganisms. To investigate the fitness of quinolone-resistant S. pneumoniae (QRSP), the relative growth efficiencies of 2 isogenic QRSP double mutants were compared with that of their fluoroquinolone-susceptible parent, EF3030, by using murine nasopharyngeal colonization and pneumonia models. Strains containing the GyrA: Ser81Phe, ParC: Ser79Phe double mutations, which are frequently seen in clinical QRSP, competed poorly with EF3030 in competitive colonization or competitive lung infections. However, they efficiently produced lung infection even in the absence of EF3030. The strain containing the GyrA: Ser81Phe, ParC: Ser79Tyr double mutations, which is seen more frequently in laboratory-derived QRSP than in clinical QRSP, demonstrated reduced nasal colonization in competitive or noncompetitive lung infections. However, the strain was equally able to cause competitive or noncompetitive lung infections as well as EF3030.

Larger numbers of pneumococci were detected in the nasal tract compared to the lung, cervical lymph nodes, and spleen 1, 2, 4, 7, 14, and 21 days after nasal challenge with Streptococcus pneumoniae strain EF3030. In this mouse model of pneumococcal carriage, peripheral S. pneumoniae pneumococcal surface adhesin A (PsaA)-specific humoral responses (immunoglobulin G2a [IgG2a] >> IgG1 = IgG2b > IgG3) were significantly higher than pneumococcal surface protein A (PspA)-specific, genetic toxoid derivative of pneumolysin (PdB)-specific, or pneumococcal surface protein C (PspC)-specific serum antibody levels. However, PspA-specific mucosal IgA antibody levels were significantly higher than those against PsaA, PdB, and PspC. In general, both PsaA- and PspA-specific lung-, cervical lymph node-, nasal tract-, and spleen-derived CD4(+) T-cell cytokine (interleukin-4, interleukin-6, granulocyte-macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha) and proliferative responses were higher than those for either PspC or PdB. Taken together, these findings suggest that PsaA- and PspA-specific mucosal responses as well as systemic humoral and T helper cell cytokine responses are predominantly yet differentially induced during pneumococcal carriage.

We characterized antibody responses to two Streptococcus pneumoniae surface proteins, PspA and PsaA, in 14 adults over 50 years of age hospitalized with invasive pneumococcal disease (IPD), and in two groups of age-matched controls (18 patients with invasive disease due to other microorganisms and 35 patients hospitalized for non infectious conditions). All patients with IPD and all control subjects had detectable antibodies to both proteins on hospital admission. Three weeks later, the geometric mean concentrations of antibodies to PspA and PsaA in IPD patients were respectively 10 and 25 times higher than on admission. In contrast, acute and convalescent antibody levels were similar in control patients with invasive diseases due to other microorganisms.

The evolution of the capsular biosynthetic (cps) locus of serogroup 6 Streptococcus pneumoniae was investigated by analyzing sequence variation within three serotype-specific cps genes from 102 serotype 6A and 6B isolates. Sequence variation within these cps genes was related to the genetic relatedness of the isolates, determined by multilocus sequence typing, and to the inferred patterns of recent evolutionary descent, explored using the eBURST algorithm. The serotype-specific cps genes had a low percent G+C, and there was a low level of sequence diversity in this region among serotype 6A and 6B isolates. There was also little sequence divergence between these serotypes, suggesting a single introduction of an ancestral cps sequence, followed by slight divergence to create serotypes 6A and 6B. A minority of serotype 6B isolates had cps sequences (class 2 sequences) that were approximately 5% divergent from those of other serotype 6B isolates (class 1 sequences) and which may have arisen by a second, more recent introduction from a related but distinct source. Expression of a serotype 6A or 6B capsule correlated perfectly with a single nonsynonymous polymorphism within wciP, the rhamnosyl transferase gene. In addition to ample evidence of the horizontal transfer of the serotype 6A and 6B cps locus into unrelated lineages, there was evidence for relatively frequent changes from serotype 6A to 6B, and vice versa, among very closely related isolates and examples of recent recombinational events between class 1 and 2 cps serogroup 6 sequences.

PsaA of Streptococcus pneumoniae, originally believed to be an adhesin, is the lipoprotein component of an Mn2+ transporter. Mutations in psaA cause deficiencies in growth, virulence, adherence, and the oxidative stress response. Immunofluorescence microscopy shows that PsaA is hidden beneath the cell wall and the polysaccharide capsule and only exposed to antibodies upon cell wall removal. A psaBC deletion mutant, expressing PsaA normally, was as deficient in adherence to Detroit 562 cells as were strains lacking PsaA. Thus, PsaA does not appear to act directly as an adhesin, but rather, psaA mutations indirectly affect this process through the disruption of Mn2+ transport. The deficiency in Mn2+ transport also causes hypersensitivity to oxidative stress from H2O2 and superoxide. In a chemically defined medium, growth of the wild-type strain was possible in the absence of Fe2+ and Mn2+ cations after a lag of about 15 h. Addition of Mn2+ alone or together with Fe2+ allowed prompt and rapid growth. In the absence of Mn2+, the addition of Fe2+ alone extended the 15-h lag phase to 25 h. Thus, while Fe2+ adversely affects the transition from lag phase to log phase, perhaps through increasing oxidative stress, this effect is relieved by the presence of Mn2+. A scavenger specific for superoxides but not those specific for hydroxyl radicals or H2O2 was able to eliminate the inhibition of growth caused by iron supplementation in the absence of Mn2+. This implies that superoxides are a key player in oxidative stress generated in the presence of iron.