Pneumococcal Capsular Polysaccharide Research Articles

Pneumococcal VncR Strain-Specifically Regulates Capsule Polysaccharide Synthesis.

Capsular polysaccharides (CPS), a major virulence factor in Streptococcus pneumoniae, become thicker during blood invasion while not during asymptomatic nasopharyngeal colonization. However, the underlying mechanism controlling this differential pneumococcal CPS regulation remain unclear. Here, we show how VncR, the response regulator of the vancomycin resistance locus (vncRS operon), regulates CPS expression in vncR mutants in three serotype (type 2, 3, and 6B) backgrounds upon exposure to serum lactoferrin (LF). Comparative analysis of CPS levels in the wild type (WT) of three strains and their isogenic vncR mutants after LF exposure revealed a strain-specific alteration in CPS production. Consistently, VncR-mediated strain-specific CPS production is correlated with pneumococcal virulence, in vivo. Electrophoretic mobility-shift assay and co-immunoprecipitation revealed an interaction between VncR and the cps promoter (cpsp) in the presence of serum. In addition, in silico analysis uncovered this protein-DNA interaction, suggesting that VncR binds with the cpsp, and recognizes the strain-specific significance of the tandem repeats in cpsp. Taken together, the interaction of VncR and cpsp after serum exposure plays an essential role in regulating differential strain-specific CPS production, which subsequently determines strain-specific systemic virulence. This study highlights how host protein LF contributes to pneumococcal VncR-mediated CPS production. As CPS plays a significant role in immune evasion, these findings suggest that drugs designed to interrupt the VncR-mediated CPS production could help to combat pneumococcal infections.

Polyamine Synthesis Effects Capsule Expression by Reduction of Precursors in Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus, Spn) colonizes the human nasopharynx asymptomatically but can cause infections such as otitis media, and invasive pneumococcal disease such as community-acquired pneumonia, meningitis, and sepsis. Although the success of Spn as a pathogen can be attributed to its ability to synthesize and regulate capsular polysaccharide (CPS) for survival in the host, the mechanisms of CPS regulation are not well-described. Recent studies from our lab demonstrate that deletion of a putative polyamine biosynthesis gene (ΔcadA) in Spn TIGR4 results in the loss of the capsule. In this study, we characterized the transcriptome and metabolome of ΔcadA and identified specific mechanisms that could explain the regulatory role of polyamines in pneumococcal CPS biosynthesis. Our data indicate that impaired polyamine synthesis impacts galactose to glucose interconversion via the Leloir pathway which limits the availability of UDP-galactose, a precursor of serotype 4 CPS, and UDP-N-acetylglucosamine (UDP-GlcNAc), a nucleotide sugar precursor that is at the intersection of CPS and peptidoglycan repeat unit biosynthesis. Reduced carbon flux through glycolysis, coupled with altered fate of glycolytic intermediates further supports impaired synthesis of UDP-GlcNAc. A significant increase in the expression of transketolases indicates a potential shift in carbon flow toward the pentose phosphate pathway (PPP). Higher PPP activity could constitute oxidative stress responses in ΔcadA which warrants further investigation. The results from this study clearly demonstrate the potential of polyamine synthesis, targeted for cancer therapy in human medicine, for the development of novel prophylactic and therapeutic strategies for treating bacterial infections.

Impact of HIV-1 infection and antigen class on T follicular helper (TFH) cell responses to pneumococcal polysaccharide-protein conjugate vaccine (PCV-13)

Abstract Pneumococcal infections are common and serious complications of HIV-1 disease. Prevention has been compromised by the limited magnitude and quality of antibody responses to T cell-independent (TI-2) pneumococcal capsular polysaccharides (PPS). The PCV-13 vaccine conjugates PPS to a T cell-dependent (TD) protein (diphtheria toxoid; DT). We investigated the differential response to PPS and DT by antibody-secreting B cells (ASC) after immunization with PCV-13 in newly-diagnosed healthy HIV+ and control adults. The numbers of PPS-specific IgG ASC increased significantly and similarly in HIV+ and controls 1 week after vaccination. However, DT-specific IgG ASC increased significantly in controls but not HIV+ subjects. To determine the cellular basis of these disparate responses to DT and PPS, we characterized the frequency and activation of T follicular helper (TFH) cells, the predominant T cell subset providing B cell help. The percentages of and intracellular IL-21 in TFH cells were unchanged with vaccination. However, expression of ICOS, which sustains TFH function and phenotype, increased significantly after vaccination among controls, but not the HIV+ group. Increases in ICOS+ TFH correlated with changes in TD DT-specific IgG ASC in controls but not in HIV+. In contrast, ICOS expression did not correlate with TI-2 PPS-specific ASC in either group. Levels of TFH differentiating factors IL-12 and IL-6 increased post vaccination only in controls. In summary, although TI-2 PPS-specific responses were similar in both groups and independent of TFH activation, TFH ICOS-expression was most closely related to responses to TD DT, which were compromised even in recently diagnosed healthy HIV+ adults.

Removal of cell wall polysaccharide in pneumococcal capsular polysaccharides by selective degradation via deamination

Pneumococcal cell wall polysaccharide (C–PS), a contaminant in pneumococcal capsular polysaccharide (Pn-PS) vaccines is degraded by mild deamination of the 4-amino-2-acetamido-2,4,6-tri-deoxy-galactose (AAT) in C–PS, which was carried out by addition of 5% aqueous sodium nitrite to a solution of polysaccharide in 5% aqueous acetic acid. Glycosidic linkage and functional groups such as O-acetates, phosphodiesters, and pyruvates were preserved under the conditions. The small fragments from degraded C–PS were removed by ultrafiltration or dialysis to provide essentially C–PS free Pn-PS. Because of the presence of AAT in its structure the deamination is not suitable for the purification of type 1 Pn-PS. Meanwhile, the mass and NMR spectroscopic analysis on the deamination products suggests that both type 1 Pn-PS and C–PS degraded following a major pathway of 5,4-hydride shift, cleavage of AAT O5-C1 bond, C1 hemiacetal formation, and its hydrolysis to release neighboring GalA- in type 1 Pn-PS and GalNAc(6-O-PCho)- in C–PS.

Comparison between a new multiplex electrochemiluminescence assay and the WHO reference enzyme-linked immunosorbent assay to measure serum antibodies against pneumococcal serotype-specific polysaccharides

BackgroundTwo electrochemiluminescence (ECL) assays were developed which, together, can simultaneously measure serum antibodies against pneumococcal capsular polysaccharides (PnPS) for 17 serotypes. The assays were validated for the 13 PnPS included in the 13-valent pneumococcal conjugate vaccine (PCV13). As recommended by the World Health Organization (WHO), we compared the ECL assays with the WHO reference enzyme-linked immunosorbent assay (ELISA) and derived a threshold corresponding to the 0.35 µg/mL threshold established for the WHO reference ELISA for the non-inferiority comparison and licensure of new PCVs against invasive pneumococcal disease. MethodsA panel of 452 serum samples from children vaccinated with one of the three licensed PCVs was assessed with the ECL assays and the WHO reference ELISA. The ECL assay threshold for the aggregated seven PnPS included in the 7-valent PCV (PCV7) and serotype-specific thresholds were determined using a receiver operating characteristics (ROC) curve-based approach and Deming regression. To evaluate concordance between the ECL assays and the WHO reference ELISA, serostatus agreement rates between both assays and geometric means of the ratios (GMRs) of concentrations obtained with both assays were calculated. ResultsThe thresholds for the seven aggregated PCV7 serotypes obtained with the ROC curve-based approach and Deming regression approximated 0.35 µg/mL (0.38 and 0.34 µg/mL, respectively). Individual thresholds for the PCV13 serotypes ranged between 0.24 and 0.51 µg/mL across both approaches. Serostatus agreement rates using a 0.35 µg/mL threshold for both assays were ≥86.9% for all PCV13 serotypes. GMRs ranged between 0.85 and 1.25 for 11/13 serotypes and were <1.29 for the two remaining serotypes. ConclusionThe ECL assays were comparable to the WHO reference ELISA and offer a sensitive, time- and serum volume-saving method to quantify serotype-specific anti-PnPS antibodies in pediatric sera. A 0.35 µg/mL threshold will be used for each PCV13 serotype to assess PCV immunogenicity in clinical trials.

The total IgM, IgA and IgG antibody responses to pneumococcal polysaccharide vaccination (Pneumovax®23) in a healthy adult population and patients diagnosed with primary immunodeficiencies

BackgroundInterpretation of the responses to the pneumococcal polysaccharide vaccine (Pneumovax®23, PPV) has proven challenging. In addition, there are few studies documenting the longevity of these responses. MethodsThe age-specific PPV IgM, IgA, IgG and IgG2 concentrations were determined pre, 4–6 weeks and 6 years post-vaccination in the serum of Prevnar®-naïve adults using VaccZyme™ pneumococcal capsular polysaccharide ELISAs. ResultsThe median pre-vaccination concentrations were; PPV IgM 53 U/mL (5–95% CI: 16-169 U/mL), IgA 23 U/mL (6-103 U/mL), IgG 41 mg/L (10-184 U/mL) and IgG2 18 mg/L (3-95 U/mL). 4–6 weeks post-vaccination there was a median 6-fold (5–95% CI: 2–24) increase in PPV IgM (median 315 U/mL (5–95% CI: 60-1133 U/mL), 18-fold (4–74) increase in IgA (369 U/mL (78-1802 U/mL)), 9-fold (2–19) increase in IgG (375 mg/L (77–1238 mg/L)) and 8-fold (1–20) increase in IgG2 (141 mg/L (25–573 mg/L)). This was significant for all isotypes in all age ranges (p < 0.0001). Six years post-vaccination median PPV concentrations were; IgM 54 U/mL (17–128), IgA 85 U/mL (19–279), IgG 148 mg/L (30–997) and IgG2 57 mg/L (9–437). The median concentrations for all ages 6 years post-vaccination were significantly elevated compared to the pre-vaccination titres for PPV IgA, IgG and IgG2 isotypes only. The PPV IgM and IgA responses were influenced by age. At 6 years post vaccination, in individuals with normal PPV IgG, 34 individuals had PPV IgM and/or IgA concentrations below the lower limit of the healthy adult ranges. We also used the healthy adult reference ranges developed in this study to assess a cohort of primary immunodeficiency (PID) patients. ConclusionThese ranges will help to provide a framework for assessment and definition of normal response to PPV, which will facilitate clinical interpretation of a deficient polysaccharide response in those suspected of antibody deficiency.

Electron Microscopy to Study the Fine Structure of the Pneumococcal Cell.

Electron microscopy allows for studying bacterial ultrastructure at high resolutions. Two types of electron microscopes are used for this purpose. The transmission electron microscope allows for access to inner bacterial ultrastructure when imaging ultrathin sections as well as cell wall-attached structures by negative staining, whereas scanning electron microscopy allows for the detection of structures on the bacterial cell surface alone or to study the interplay between pneumococci and their host cells. This chapter deals with recommendations for well-adapted methodologies to examine pneumococcal ultrastructure in detail. Especially, we focus on the preservation of the pneumococcal capsular polysaccharide, which represents an important virulence factor of pneumococci. Since capsules are highly hydrated structures, the introduction of a new fixation protocol involving lysine acetate, ruthenium red, and osmium (LRR fixation) results in a very well-preserved capsular structure in such a way that the amount of capsular material bound on the bacterial surface can be compared within different serotypes. In our method, capsular ultrastructure is preserved without the need for serotype-specific antibodies, which have been used in other studies to preserve the pneumococcal capsule. In addition, the new LRR fixation allows for studying the presence or absence of capsular material during adhesion and invasion of pneumococci on epithelial or endothelial host cells in cell culture experiments.

Semisynthetic glycoconjugate based on dual role protein/PsaA as a pneumococcal vaccine

Pneumococcal infections remain a major public health concern worldwide. The currently available vaccines in the market are based on pneumococcal capsular polysaccharides but they still need to be improved to secure an optimal coverage notably in population at risk. To circumvent this, association of virulence pneumococcal proteins to the polysaccharide valencies has been proposed with the hope to observe an additive - if not synergistic - protective effect. Along this line, the use of the highly conserved and ubiquitous pneumococcal surface adhesin A (PsaA) as a protein carrier for a synthetic pneumococcal oligosaccharide is demonstrated herein for the first time. A tetrasaccharide mimicking functional antigenic determinants from the S. pneumoniae serotype 14 capsular polysaccharide (Pn14TS) was chemically synthesised. The mature PsaA (mPsaA) was expressed in E. coli and purified using affinity chromatography. The Pn14PS was conjugated to mPsaA using maleimide-thiol coupling chemistry to obtain mPsaA-Pn14PS conjugate (protein/sugar molar ratio: 1/5.4). The mPsaA retained the structural conformation after the conjugation and lyophilisation. The prepared glycoconjugate adjuvanted with α-galactosylceramide, a potent activator of invariant Natural Killer T cells, was tested in mice for its immunological response upon subcutaneous injection in comparison with mPsaA alone and a model BSA conjugate (BSA-Pn14PS, used here as a control). Mice immunised with the mPsaA-Pn14TS produced a robust IgG response against mPsaA and against the capsular polysaccharide from pneumococcal serotype 14. These data provide the basis for novel pneumococcal vaccine development.

642. B- and T-Cell Responses to Pneumococcal Polysaccharide and Protein Vaccine Antigens in Recently Diagnosed HIV-1-Infected Patients

BackgroundsPrevention of serious HIV-1-associated pneumococcal infections may be compromised by the limited magnitude and function of vaccine-induced antibodies. Responses to the T-independent pneumococcal capsular polysaccharide (PPS) + T-dependent diphtheria toxoid (DT) protein conjugate vaccine (PCV-13) may be influenced by CD4+ T follicular helper (TFH) cells which provide specific help for B-cell differentiation.MethodsWe immunized 22 control and 19 newly diagnosed HIV-1-infected adults (median 610 CD4+ T cells/µL (range: 139–1,408) and 69,316 plasma HIV RNA (range 232-806,936) on ART for 1–4 months with PCV13. We measured (i) PPS-specific antibody-secreting cells (ASC) by ELISPOT at Weeks 0 and 1, (ii) serum IgG to 11 PPS serotypes (ST) by multiplex ELISA and (iii) titers of opsonophagocytosis (OP) for four STs at Weeks 0 and 8, and (iv) numbers and activation (ICOS expression) of circulating TFH cells by flow cytometry at Weeks 0 and 1. Values were compared by ANOVA, paired and unpaired t and Mann–Whitney tests.ResultsThe number of PPS-specific IgG, IgM and IgA ASC increased significantly from Weeks 0 to 1 post-PCV13 and to similar magnitude in both Controls and HIV+ subjects, returning to baseline by Week 8. Levels of serum PPS-specific IgG increased significantly from Weeks 0 to 8 for 10/11 vs. 7/11 ST in controls and HIV+ subjects, respectively (P = NS), and to comparable levels. Similarly, OP titers increased significantly and similarly to each of four STs in both groups from Weeks 0 to 8. In contrast, although DT-specific IgG ASC increased from Weeks 0 to 1 in HIV+ and controls, these values were lower among HIV-1+ adults (P = .001). Consistent with these limited responses, a key regulatory molecule on TFH cells, elicited largely by T-dependent antigens (DT), was upregulated on cells from Control but not HIV+ at Week 1. Moreover, levels of IL-12, which drives TFH differentiation, were also lower among HIV-1+ at Week 1. Conclusion. Humoral responses to PPS are largely intact (ASC, serum IgG and killing function) with recently diagnosed HIV-1 infection, highlighting the importance of early HIV-1 recognition. That responses to T-dependent DT and TFH activation are more limited, even with high CD4+ counts and ART, suggests a more rapid and perhaps more recalcitrant HIV-1-associated T-cell defect.Disclosures All authors: No reported disclosures.

Engineering a Next-Generation Glycoconjugate-Like Streptococcus pneumoniae Vaccine.

We detail the development of a next-generation Streptococcus pneumoniae liposomal encapsulation of polysaccharides (LEPS) vaccine, with design characteristics geared toward best-in-class efficacy. The first generation LEPS vaccine, which contained 20 encapsulated pneumococcal capsular polysaccharides (CPSs) and two surface-displayed virulence-associated proteins (GlpO and PncO), enabling prophylactic potency against 70+ serotypes of Streptococcus pneumoniae (the causative agent of pneumococcal disease), was rationally redesigned for advanced clinical readiness and best-in-class coverage. In doing so, the virulent-specific GlpO protein antigen was removed from the final formulation due to off-target immunogenicity toward bacterial species within the human microbiome, while directed protection was maintained by increasing the dose of PncO from 17 to 68 μg. LEPS formulation parameters also readily facilitated an increase in CPS valency (to a total of 24) and systematic variation in protein-liposome attachment mechanisms in anticipation of clinical translation. An additional safety assessment study demonstrated that LEPS does not exhibit appreciable toxicological effects even when administered at ten times the effective dose. In summary, this new design offers the broadest, safest, and most-complete protection while maintaining desirable glycoconjugate-like features, positioning the LEPS vaccine platform for clinical success and a global health impact.

Immunogenicity and safety of 11- and 12-valent pneumococcal non-typeable Haemophilus influenzae protein D-conjugate vaccines (11vPHiD-CV, 12vPHiD-CV) in infants: Results from a phase II, randomised, multicentre study

BackgroundWe assessed 2 investigational 11- and 12-valent vaccines, containing capsular polysaccharides of 10 serotypes as in the pneumococcal non-typeable Haemophilus influenzae protein D-conjugate vaccine (PHiD-CV) and CRM197-conjugated capsular polysaccharides of serotypes 19A (11-valent) or 19A and 6A (12-valent). MethodsIn this phase II, partially-blind, multicentre study (NCT01204658), healthy infants were randomised (1:1:1:1) to receive 11vPHiD-CV, 12vPHiD-CV, PHiD-CV, or 13-valent CRM197-conjugate pneumococcal vaccine (PCV13), at 2, 3, and 4 (primary series), and 12–15 months of age (booster dose), co-administered with DTPa-HBV-IPV/Hib. Confirmatory objectives assessed non-inferiority of investigational vaccines to comparators (PHiD-CV for common serotypes; PCV13 for 19A and 6A), in terms of percentage of infants with pneumococcal antibody concentrations ≥0.2 μg/mL and antibody geometric mean concentrations, post-primary vaccination. Reactogenicity and safety were assessed. Results951 children received ≥1 primary dose, 919 a booster dose. Pre-defined immunological non-inferiority criteria were met simultaneously for 9/11 11vPHiD-CV serotypes (all except 23F and 19A) and 10/12 12vPHiD-CV serotypes (all except 19A and 6A); thus, non-inferiority objectives were reached. For each PHiD-CV serotype, percentages of children with antibody concentrations ≥0.2 µg/mL were ≥96.7% post-primary (except 6B [≥75.2%] and 23F [≥81.1%]), and ≥98.1% post-booster vaccination. For each PHiD-CV serotype except serotype 1, ≥81.0% and ≥93.9% of children had opsonophagocytic activity titres ≥8, post-primary and booster vaccination. AEs incidence was similar across all groups. SAEs were reported for 117 children (29 in the 11vPHiD-CV group, 26 in the 12vPHiD-CV group, 38 in the PHiD-CV group and 24 in the PCV13 group); 4 SAEs were considered vaccination-related. No fatal events were recorded. ConclusionAddition of 19A and 6A CRM197-conjugates did not alter immunogenicity of the PHiD-CV conjugates; for both investigational vaccines post-booster immune responses to 10 common serotypes appeared similar to those elicited by PHiD-CV. Safety and reactogenicity profiles of the investigational vaccines were comparable to PHiD-CV.Clinical trial registry: NCT01204658.

Enzymatic Hydrolysis of Pneumococcal Capsular Polysaccharide Renders the Bacterium Vulnerable to Host Defense.

Despite a century of investigation, Streptococcus pneumoniae remains a major human pathogen, causing a number of diseases, such as pneumonia, meningitis, and otitis media. Like many encapsulated pathogens, the capsular polysaccharide (CPS) of S. pneumoniae is a critical component for colonization and virulence in mammalian hosts. This study aimed to evaluate the protective role of a glycoside hydrolase, Pn3Pase, targeting the CPS of type 3 S. pneumoniae, which is one of the most virulent serotypes. We have assessed the ability of Pn3Pase to degrade the capsule on a live type 3 strain. Through in vitro assays, we observed that Pn3Pase treatment increases the bacterium's susceptibility to phagocytosis by macrophages and complement-mediated killing by neutrophils. We have demonstrated that in vivo Pn3Pase treatment reduces nasopharyngeal colonization and protects mice from sepsis caused by type 3 S. pneumoniae Due to the increasing shifts in serotype distribution, the rise in drug-resistant strains, and poor immune responses to vaccine-included serotypes, it is necessary to investigate approaches to combat pneumococcal infections. This study evaluates the interaction of pneumococcal CPS with the host at molecular, cellular, and systemic levels and offers an alternative therapeutic approach for diseases caused by S. pneumoniae through enzymatic hydrolysis of the CPS.

A Capsular Polysaccharide-Specific Antibody Alters Streptococcus pneumoniae Gene Expression during Nasopharyngeal Colonization of Mice.

Pneumococcal conjugate vaccines (PCV) elicit opsonophagocytic (opsonic) antibodies to pneumococcal capsular polysaccharides (PPS) and reduce nasopharyngeal (NP) colonization by vaccine-included Streptococcus pneumoniae serotypes. However, nonopsonic antibodies may also be important for protection against pneumococcal disease. For example, 1E2, a mouse IgG1 monoclonal antibody (MAb) to the serotype 3 (ST3) PPS (PPS3), reduced ST3 NP colonization in mice and altered ST3 gene expression in vitro Here, we determined whether 1E2 affects ST3 gene expression in vivo during colonization of mice by performing RNA sequencing on NP lavage fluid from ST3-infected mice treated with 1E2, a control MAb, or phosphate-buffered saline. Compared to the results for the controls, 1E2 significantly altered the expression of over 50 genes. It increased the expression of the piuBCDA operon, which encodes an iron uptake system, and decreased the expression of dpr, which encodes a protein critical for resistance to oxidative stress. 1E2-mediated effects on ST3 in vivo required divalent binding, as Fab fragments did not reduce NP colonization or alter ST3 gene expression. In vitro, 1E2 induced dose-dependent ST3 growth arrest and altered piuB and dpr expression, whereas an opsonic PPS3 MAb, 5F6, did not. 1E2-treated bacteria were more sensitive to hydrogen peroxide and the iron-requiring antibiotic streptonigrin, suggesting that 1E2 may increase iron import and enhance sensitivity to oxidative stress. Finally, 1E2 also induced rapid capsule shedding in vitro, suggesting that this may initiate 1E2-induced changes in sensitivity to oxidative stress and gene expression. Our data reveal a novel mechanism of direct, antibody-mediated antibacterial activity that could inform new directions in antipneumococcal therapy and vaccine development.

Comparison of serological assays using pneumococcal proteins or polysaccharides for detection of Streptococcus pneumoniae infection in children with community-acquired pneumonia

The aim of this study was to compare the results of serological assays using pneumococcal proteins or polysaccharides for the detection of pneumococcal infection in childhood pneumonia. Serological assays measured IgG against eight pneumococcal proteins (Ply,CbpA,PspA1,PspA2,PcpA,PhtD,StkP-C,PcsB-N), C-polysaccharide [in the whole study population, n = 183], or 19 pneumococcal capsular polysaccharides (1,2,4,5,6B,7F,8,9 V,10A,11A,12F,14,15B,17F,18C,19F,20,23F,33F) [only in a subgroup of patients, n = 53] in paired serum samples of children aged <5 years-old hospitalized with clinical and radiological diagnosis of community-acquired pneumonia. We also performed an inhibition of binding test with the anti-capsular polysaccharide assay in order to confirm the specificity of the antibody responses detected. Invasive pneumococcal pneumonia was investigated by blood culture and PCR (ply-primer). Among 183 children, the anti-protein assay detected antibody response in 77/183(42.1%) patients and the anti-C-polysaccharide assay in 28/183(15.3%) patients. In a subgroup of 53 children, the anti-protein assay detected response in 32/53(60.4%) patients, the anti-C-polysaccharide assay in 11/53(20.8%) patients, and the anti-capsular polysaccharide in 25/53(47.2%) patients. Simultaneous antibody responses against ≥2 different capsular polysaccharides were detected in 11/53(20.8%) patients and this finding could not be explained by cross-reactivity between different serotypes. Among 13 patients with invasive pneumococcal pneumonia, the sensitivity of the anti-protein assay was 92.3%(12/13), of the anti-C-polysaccharide assay 30.8%(4/13), and of the anti-capsular polysaccharide assay 46.2%(6/13). The serological assay using pneumococcal proteins is more sensitive for the detection of pneumococcal infection in children with pneumonia than the assay using pneumococcal polysaccharides. Future studies on childhood pneumonia aetiology should consider applying serological assays using pneumococcal proteins.

Anti-protein immunoglobulin M responses to pneumococcus are not associated with aging

BackgroundThe incidence of community-acquired pneumonia and lower respiratory tract infection rises considerably in later life. Immunoglobulin M (IgM) antibody levels to pneumococcal capsular polysaccharide are known to decrease with age; however, whether levels of IgM antibody to pneumococcal proteins are subject to the same decline has not yet been investigated.MethodsThis study measured serum levels and binding capacity of IgM antibody specific to the pneumococcal surface protein A (PspA) and an unencapsulated pneumococcal strain in serum isolated from hospital patients aged < 60 and ≥ 60, with and without lower respiratory tract infection. A group of young healthy volunteers was used as a comparator to represent adults at very low risk of pneumococcal pneumonia. IgM serum antibody levels were measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry was performed to assess IgM binding capacity. Linear regression and one-way analysis of variance (ANOVA) tests were used to analyse the results.ResultsLevels and binding capacity of IgM antibody to PspA and the unencapsulated pneumococcal strain were unchanged with age.ConclusionsThese findings suggest that protein-based pneumococcal vaccines may provide protective immunity in the elderly.Trial registrationThe LRTI trial (LRTI and control groups) was approved by the National Health Service Research Ethics Committee in October 2013 (12/NW/0713). Recruitment opened in January 2013 and was completed in July 2013. Healthy volunteer samples were taken from the EHPC dose-ranging and reproducibility trial, approved by the same Research Ethics Committee in October 2011 (11/NW/0592). Recruitment for this study ran from October 2011 until December 2012. LRTI trial: (NCT01861184), EHPC dose-ranging and reproducibility trial: (ISRCTN85403723).

Gut Microbiota Contributes to Resistance Against Pneumococcal Pneumonia in Immunodeficient Rag-/- Mice.

Streptococcus pneumoniae causes infection-related mortality worldwide. Immunocompromised individuals, including young children, the elderly, and those with immunodeficiency, are especially vulnerable, yet little is known regarding S. pneumoniae-related pathogenesis and protection in immunocompromised hosts. Recently, strong interest has emerged in the gut microbiota's impact on lung diseases, or the “gut-lung axis.” However, the mechanisms of gut microbiota protection against gut-distal lung diseases like pneumonia remain unclear. We investigated the role of the gut commensal, segmented filamentous bacteria (SFB), against pneumococcal pneumonia in immunocompetent and immunocompromised mouse models. For the latter, we chose the Rag−/− model, with adaptive immune deficiency. Immunocompetent adaptive protection against S. pneumoniae infection is based on antibodies against pneumococcal capsular polysaccharides, prototypical T cell independent-II (TI-II) antigens. Although SFB colonization enhanced TI-II antibodies in C57BL/6 mice, our data suggest that SFB did not further protect these immunocompetent animals. Indeed, basal B cell activity in hosts without SFB is sufficient for essential protection against S. pneumoniae. However, in immunocompromised Rag−/− mice, we demonstrate a gut-lung axis of communication, as SFB influenced lung protection by regulating innate immunity. Neutrophil resolution is crucial to recovery, since an unchecked neutrophil response causes severe tissue damage. We found no early neutrophil recruitment differences between hosts with or without SFB; however, we observed a significant drop in lung neutrophils in the resolution phase of S. pneumoniae infection, which corresponded with lower CD47 expression, a molecule that inhibits phagocytosis of apoptotic cells, in SFB-colonized Rag−/− mice. SFB promoted a shift in lung neutrophil phenotype from inflammatory neutrophils expressing high levels of CD18 and low levels of CD62L, to pro-resolution neutrophils with low CD18 and high CD62L. Blocking CD47 in SFB(−) mice increased pro-resolution neutrophils, suggesting CD47 down-regulation may be one neutrophil-modulating mechanism SFB utilizes. The SFB-induced lung neutrophil phenotype remained similar with heat-inactivated S. pneumoniae treatment, indicating these SFB-induced changes in neutrophil phenotype during the resolution phase are not simply secondary to better bacterial clearance in SFB(+) than SFB(−) mice. Together, these data demonstrate that the gut commensal SFB may provide much-needed protection in immunocompromised hosts in part by promoting neutrophil resolution post lung infection.

Relationship of specific IgM responses with infection and lymphoproliferative states in common variable immunodeficiency

While common variable immunodeficiency (CVID) markedly impairs IgA and IgG responses, IgM production is retained in some patients. This retained IgM production is linked with protection against infection as well as susceptibility to lymphoproliferative complications. To gain more insight into the role of IgM in CVID, we examined whether antigen-specific IgM levels differentiated patients with history of pneumonia or those with lymphoproliferative complications. Serum IgM recognizing bacterial (capsular polysaccharides, cell wall polysaccharides, flagellin, and lipopolysaccharide) or natural IgM (Forssmann antigen, phosphocholine) antigens were measured by enzyme-linked immunosorbent assay (ELISA). Patients meeting consensus guidelines for CVID were included in this study. Retrospective chart review was utilized to determine history of infections and other medical complications. This study was approved by the institutional review board of the Icahn School of Medicine at Mount Sinai. Compared to CVID patients without history of pneumonia, those with history of pneumonia had significantly lower levels of IgM recognizing bacterial cell wall antigens and flagellin, but interestingly had similar levels of IgM recognizing pneumococcal capsular polysaccharides. In contrast, significant increase of serum IgM recognizing lipopolysaccharide was found in CVID patients with lymphoproliferative complications compared to those without. Levels of serum IgM recognizing specific antigens may differentiate CVID patients with history of lower respiratory tract infections or lymphoproliferative complications. Furthermore, the investigation of antigen-specific IgM may help to explain previous investigations linking normal or elevated IgM levels to both protection against infection and susceptibility to lymphoproliferation in CVID.

Immune-potentiation of Pneumococcal Capsular Polysaccharide Antigen using Albumin Microparticles

Microparticles (MPs) offer several advantages as unique vaccine delivery system, including the ease to manufacture, targeted delivery of antigen payload, sustained antigen release and possible role as an immuneadjuvant. In this study, we evaluated albumin matrix for pneumococcal (Pnc) serotype specific capsular polysaccharide (PS) antigen MPs. Microencapsulation of Pnc PS was successful with a product yield of >72%. The MP size, 1-5 μm, and negative zeta potential (-26.5 mV) were optimized to ensure effective -uptake and presentation of Pnc PS antigens to immune cells. In mice, ST 19F and 23F MPs exhibited >10-fold increase (P<0.01) in ST specific IgG response over PS in solution given with or without alum. Relatively higher immune response was observed for ST 6B MPs when compared to PS solution; however, ST6B PS solution along with alum resulted in an overall higher response when compared to ST6B MPs. Microencapsulation may offer a simple and effective mechanism for the immune-enhancement of poorly immunogenic antigens such as Pnc PS.

Genetic and structural elucidation of capsular polysaccharides from Streptococcus pneumoniae serotype 23A and 23B, and comparison to serotype 23F

Streptococcus pneumoniae is a globally important encapsulated human pathogen with approximately 100 different serotypes recognized. Serogroup 23 consists of serotype 23F, present in licensed vaccines, and emerging serotypes 23A and 23B. Here, we report the previously unknown structures of the pneumococcal capsular polysaccharides serotype 23A and 23B determined using genetic analysis, NMR spectroscopy, composition and linkage analysis and Smith degradation (of polysaccharide 23A). The structure of the serotype 23A capsular polysaccharide is: →4)-β-D-Glcp-(1→3)-[[α-L-Rhap-(1→2)]-[Gro-(2→P→3)]-β-D-Galp-(1→4)]-β-L-Rhap-(1→. This structure differs from polysaccharide 23F as it features a disaccharide backbone and the di-substituted β-Gal is linked to β-Rha as a side chain. This is due to the different polymerization position catalysed by the unusually divergent repeat unit polymerase Wzy in the 23A cps biosynthesis locus. Steric crowding in 23A, confirmed by molecular models, causes the NMR signal for H-1 of the di-substituted 2,3-β-Gal to resonate in the α-anomeric region. The structure of the serotype 23B capsular polysaccharide is the same as 23F, but without the terminal α-Rha: →4)-β-D-Glcp-(1→4)-[Gro-(2→P→3)]-β-D-Galp-(1→4)-β-L-Rhap-(1→. The immunodominant terminal α-Rha of 23F is more sterically crowded in 23A and absent in 23B. This may explain the reported typing cross reactions for serotype 23F: slight with 23A and none with 23B.

Concentrations of Pneumococcal IgA and IgM are compromised in some individuals with antibody deficiencies

ABSTRACTThe response to pneumococcal vaccination is assessed by measurement of antigen specific IgG only and is compromised in a number of antibody deficiencies. We measured the concentrations of Pneumococcal IgA and IgM in individuals with both normal and abnormal pneumococcal capsular polysaccharide (PCP) IgG concentrations. A higher number of individuals had abnormal pre-vaccination IgA and IgM concentrations below the lower limit of the normal range compared to the control group. Post vaccination a lower number of individuals had IgA and IgM concentrations below the upper limit of the normal range compared to the control group. Non responders had a higher percentage of individuals with a prior history of infection. In addition, individuals with a history of prior infection had lower pre- and post-vaccination concentrations of PCP IgG, IgA, and IgM. Post-vaccination IgA and IgM concentrations identified four groups of responses which correlated with prior history of infection. A higher percentage of individuals with abnormal PCP IgA and IgM concentrations had a history of prior infection compared to the percentage of individuals with normal concentrations. In individuals with an antibody deficiency, measurement of Pneumococcal IgA and IgM correlates with the number of individuals with prior history of infection.