Pneumococcal Capsular Polysaccharide Serotype Research Articles

Sex Influences Age-Related Changes in Natural Antibodies and CD5+ B-1 Cells.

Natural Abs are primarily produced by B-1 cells and are essential for protection against Streptococcus pneumoniae The incidence and mortality rate for pneumococcal infection increases dramatically after age 65, disproportionately affecting males in both human and murine systems. To date, there is a significant gap in our understanding of the relationship among sex, aging, natural IgM efficacy, and the natural IgM repertoire. Our investigation demonstrates that the protective capacity of serum IgM against pneumococcal infection is maintained in IgM obtained from aged female mice but absent in IgM from aged male mice. To understand this difference in protective capacity, we examined serum Ig, discovering that the protective change was not associated with shifts in levels of phosphorylcholine (PC)- or pneumococcal capsular polysaccharide serotype 3-specific IgM. Interestingly, we observed that aged females have an increase in the total number of CD5+ B-1 cells, higher serum IL-5 levels, and a larger percentage of aged female CD5+ B-1 cells that express CD86 as compared with aged males. Furthermore, single-cell IgM repertoire analysis from peritoneal PC+, splenic PC+, and bone marrow CD5+ B-1 cell subsets demonstrated greater diversity with age and a higher level of germline status in female mice than previously observed in studies of aged male mice. Aged female CD5+ B-1 cells also expressed higher levels of transcripts associated with cell activity and self-renewal, such as Nanog and Hmga2 Taken together, these data indicate that females maintain a more diverse and active CD5+ B-1 cell pool and natural IgM repertoire, which has implications for sex-related susceptibility to infection and disease.

Development of a pneumococcal conjugate vaccine based on chemical conjugation of polysaccharide serotype 6B to PspA

The use of conjugate vaccines remains an effective intervention to prevent pneumococcal diseases. In order to expand vaccine coverage, the inclusion of pneumococcal proteins as carriers is a propitious alternative that has been explored over the past few years. In this study, pneumococcal surface protein A (PspA) clade 1, family 1 (PspA1) and clade 3, family 2 (PspA3) were used as carrier proteins for pneumococcal capsular polysaccharide serotype 6B (Ps6B). Employing an improved reductive amination chemistry, 50% of Ps6B was incorporated to each protein, PspA1 and PspA3. The effect of chemical modifications in Ps6B and PspA was assessed by an antigenicity assay and circular dichroism, respectively. Fragmentation and oxidation decreased the antigenicity of Ps6B while conjugation improved antigenicity. In the same manner, introduction of adipic acid dihydrazide (ADH) reduced PspA secondary structure content, which was partially restored after conjugation. Immunization of Ps6B-PspA1 and Ps6B-PspA3 conjugates in mice induced specific IgG antibodies against the Ps6B and the protein; and anti-PspA antibodies had functional activity against two pneumococcal strains with different serotypes. These results suggest that chemical coupling between Ps6B and PspA did not affect antigenic epitopes and support the further development of PspA as a carrier protein in pneumococcal conjugate vaccines to provide broader protection.

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.

Serotypes and genotypes of Streptococcus pneumoniae isolates from Trinidad and Tobago.

SUMMARYObjectivesThere are currently 94 known pneumococcal capsular polysaccharide serotypes and their prevalence differs by geographic region and the period studied. Streptococcus pneumoniae infections have been diagnosed clinically in Trinidad and Tobago and other Caribbean countries, however data on the serotype and sequence type distributions in this country are limited. The objective of this study was to determine serotypes and multilocus sequence types (MLSTs) of invasive and non-invasive pneumococcal isolates from Trinidad and Tobago.MethodsNinety-eight pneumococcal isolates from several regional hospitals in the country were analyzed using both standard microbiological methods and molecular analysis. These isolates included invasive (n = 83) and selected non-invasive (n = 15) strains recovered before (n = 25) and after (n = 73) the introduction of the pneumococcal conjugate vaccine.ResultsMore than half of the isolates (54.1%) were recovered from children under 15 years of age, with the largest proportion being from children under 2 years of age (24.5%). The most prevalent serotypes were 19F (18.4%), 6B (15.3%), 23F (14.3%), 3 (11.2%), 19A (6.1%), 6A (5.1%), 14 (5.1%), and 9V (4.1%). The most common serotype/MLST combinations were 6B/ST138 (n = 10, 10.2%), 3/ST180 (n = 5, 5.1%), 23F/ST629 (n = 5, 5.1%), 19F/ST8398 (n = 4, 4.1%), and three each of 6B/ST145, 14/9V/ST156, 9V/ST162, 19A/320, and 3/ST10440.ConclusionsThis report provides the first glimpse of the prevailing pneumococcal sequence types in the country. Most of the isolates represented serotypes in the 10-valent (61.2% of isolates) and 13-valent (83.7%) pneumococcal conjugate vaccines. A detailed population study is warranted to fully determine the circulating pneumococcal sequence types. Furthermore, the implementation of an effective and continuous surveillance system in Trinidad and Tobago is paramount to monitor vaccine impact.

Identification of the common antigenic determinant shared by Streptococcus pneumoniae serotypes 33A, 35A, and 20 capsular polysaccharides

In order to better understand cross-reactions of serogroup 33 polysaccharides and the typing sera, the structure of pneumococcal capsular polysaccharide serotype 33A was elucidated. Serotype 33A has been shown to have an identical polysaccharide backbone as that of serotype 33F, with two additional sites of O-acetylation at C5, and C6 of the 3-β-Galf residue in serotype 33A. This finding is consistent with the presence of an additional functional acetyltransferase gene (wcjE) in the cps biosynthetic locus of serotype 33A compared to 33F. The identical polysaccharide backbone with at least one common O-acetylation site (C2 of 5-β-Galf) shared by serotype 33A and 33F polysaccharides is proposed to be the epitope recognized by typing serum 33b. In addition, a 5,6-di-O-acetylated →3)-β-d-Galf5,6Ac-(1→3)-β-d-Glcp-(1→ disaccharide unit, a common structural motif present in serotypes 33A, 20, and 35A polysaccharides, is proposed to be the antigenic determinant recognized by typing serum 20b.

Differential Idiotype Utilization for the In Vivo Type 14 Capsular Polysaccharide-Specific Ig Responses to Intact Streptococcus pneumoniae versus a Pneumococcal Conjugate Vaccine

Murine IgG responses specific for the capsular polysaccharide (pneumococcal capsular polysaccharide serotype 14; PPS14) of Streptococcus pneumoniae type 14 (Pn14), induced in response to intact Pn14 or a PPS14-protein conjugate, are both dependent on CD4(+) T cell help but appear to use marginal zone versus follicular B cells, respectively. In this study, we identify an idiotype (44.1-Id) that dominates the PPS14-specific IgG, but not IgM, responses to intact Pn14, isolated PPS14, and Group B Streptococcus (strain COH1-11) expressing capsular polysaccharide structurally identical to PPS14. The 44.1-Id, however, is not expressed in the repertoire of natural PPS14-specific Abs. In distinct contrast, PPS14-specific IgG responses to a soluble PPS14-protein conjugate exhibit minimal usage of the 44.1-Id, although significant 44.1-Id expression is elicited in response to conjugate attached to particles. The 44.1-Id elicited in response to intact Pn14 was expressed in similar proportions among all four IgG subclasses during both the primary and secondary responses. The 44.1-Id usage was linked to the Igh(a), but not Igh(b), allotype and was associated with induction of relatively high total PPS14-specific IgG responses. In contrast to PPS14-protein conjugate, avidity maturation of the 44.1-Id-dominant PPS14-specific IgG responses was limited, even during the highly boosted T cell-dependent PPS14-specific secondary responses to COH1-11. These results indicate that different antigenic forms of the same capsular polysaccharide can recruit distinct B cell clones expressing characteristic idiotypes under genetic control and suggest that the 44.1-Id is derived from marginal zone B cells.

Humoral immune response of a pneumococcal conjugate vaccine: Capsular polysaccharide serotype 14—Lysine modified PspA

Polysaccharide-protein conjugates are so far the current antigens used for pneumococcal vaccines for children under 2 years of age. In this study, pneumococcal surface protein A (PspA) was used as a carrier protein for pneumococcal capsular polysaccharide serotype 14 as an alternative to broaden the vaccine coverage. PspA was modified by reductive amination with formaldehyde in order to improve the specificity of the reaction between protein and polysaccharide, inhibiting polymerization and the gel formation reaction. In the synthesis process, the currently used activator, 1-[3-(dimethylamine)propyl]-3-ethylcarbodiimide hydrochloride (EDAC) was substituted for 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). BALB/c mice were immunized with either the PS14-mPspA conjugate or the co-administered components in a three dose regimen and sera from the immunized animals were assayed for immunity induced against both antigens: PS14 and mPspA. Modification of more than 70% of lysine residues from PspA (mPspA) did not interfere in the immune response as evaluated by the anti-PspA titer and C3 complement deposition assay. Sera of mice immunized with conjugated PS14-mPspA showed similar IgG titers, avidity and isotype profile as compared to controls immunized with PspA or mPspA alone. The complement deposition was higher in the sera of mice immunized with the conjugate vaccine and the opsonophagocytic activity was similar for both sera. Conjugation improved the immune response against PS14. The anti PS14 IgG titer was higher in sera of mice immunized with the conjugate than with co-administered antigens and presented an increased avidity index, induction of a predominant IgG1 isotype and increased complement deposition on a bacteria with a surface serotype 14. These results strongly support the use of PspA as carrier in a conjugate vaccine where both components act as antigens.

Adenoidectomy and nasopharyngeal carriage of Streptococcus pneumoniae in young children

ObjectiveThe effect of adenoidectomy on nasopharyngeal colonisation of pathogens has not previously been evaluated. The authors studied the effect of adenoidectomy on nasopharyngeal colonisation by bacteria causing otitis media and...

Peptide mimics of two pneumococcal capsular polysaccharide serotypes (6B and 9V) protect mice from a lethal challenge with Streptococcus pneumoniae

Anti-polysaccharide immunity is a key facet of protection against several bacterial pathogens. Problems exist with current polysaccharide vaccines and alternative strategies that deliver a protective response are needed. We have identified immunological peptide mimics of type 6B and 9V pneumococcal capsular polysaccharides that could be used as vaccine antigens. Peptides mimicking antigenic properties of serotype 6B capsular polysaccharide were obtained from a phage-displayed peptide library expressing dodecameric peptides, using a human monoclonal antibody (Db3G9). A murine monoclonal antibody (206, F-5) against the serotype 9V capsular polysaccharide identified three peptide mimotopes from the dodecameric peptide library and one from a random pentadecameric peptide library. In ELISA, binding of 206, F-5 and Db3G9 to phage displaying the selected mimotopes was significantly inhibited by type-specific pneumococcal polysaccharide. Peptides were conjugated to keyhole limpet haemocyanin and were used to immunise mice. Two peptides, MP13 and MP7, induced specific anti-6B and 9V polysaccharide antibodies, respectively. Mice immunised with MP7-keyhole limpet hemocyanin or MP13-keyhole limpet hemocyanin conjugate were significantly and specifically protected against a lethal challenge with pneumococci of the appropriate serotype. This study provides strong in vivo evidence that peptide mimics are alternatives to polysaccharide vaccines.

Competitive Inhibition Flow Analysis Assay for the Non-Culture-Based Detection and Serotyping of Pneumococcal Capsular Polysaccharide

Traditional confirmation procedures for the identification of a pneumococcal serotype require an isolate. Non-culture-based confirmation protocols are available. Some of these confirm only the presence of pneumococci, and others are capable of identifying a limited number of serotypes. The increased use of pneumococcal polysaccharide and conjugate vaccines, especially in high-risk patient groups, and the likely increase in the number of serotypes included in future versions of the conjugate vaccines have necessitated the need for improved enhanced surveillance in order to assess their impact on public health. Since 2006, a multiplexed assay has been used at the Health Protection Agency of the United Kingdom for the detection of 14 pneumococcal serotypes which requires pneumococcal serotype-specific monoclonal antibodies (MAbs). We have developed a microsphere competitive inhibition method capable of detecting 23 pneumococcal capsular polysaccharide serotypes in cerebrospinal fluid (CSF) and urine and serotyping pneumococcal suspensions, utilizing an international reference serum, 89-SF. The assay was shown to be reproducible and specific for homologous polysaccharide. Validation of the assay was performed with a selection of MAbs specific for pneumococcal capsular polysaccharide serotypes, which confirmed the specificity of the assay. Analysis of pneumolysin PCR-positive CSF samples in the competitive inhibition assay determined a serotype for 89% of the samples. The assay developed here is well suited to large-scale epidemiologic studies because the assay is simple, robust, and rapid and utilizes readily available resources.

A Human Monoclonal Immunoglobulin M Reduces Bacteremia and Inflammation in a Mouse Model of Systemic Pneumococcal Infection

Antibody-based approaches to pneumococcal disease may hold promise for immunocompromised patients in whom vaccines are less immunogenic and/or in the context of antimicrobial resistance. Antibody-mediated protection against experimental pneumococcal pneumonia has been shown to depend on immunoregulation, but the relationship between antibody and protection against pneumococcal sepsis and immunoregulation has not been examined. Similarly, the requirement for B and T cells for antibody efficacy is not known. In this study, we determined the efficacy of the human pneumococcal capsular polysaccharide serotype 3-specific antibody, A7 (immunoglobulin M [IgM]), in secretory IgM (sIgM)(-/-), CD4(-/-), CD8(-/-), muMT(-/-), and SCID mice and investigated its effect on cytokine and chemokine expression in sera and spleens from mice with intact cellular immunity. A7 is known to be protective against systemic infection with serotype 3 and to require complement for efficacy. Compared to that of an isotype control antibody, A7 administration prolonged the survival of mice of each immunodeficient strain and was associated with a significant reduction in CFU in blood, lung, and spleen samples and a significantly reduced level of keratinocyte-derived chemokine (KC), interleukin-6 (IL-6), and macrophage inflammatory protein-2 (MIP-2) expression in normal and sIgM(-/-) mice. Studies with mice treated with penicillin revealed similar reductions in CFU and similar levels of IL-6, KC, or MIP-2 expression in A7- and penicillin-treated mice. These findings demonstrate that natural IgM and B and T cells are dispensable for A7-mediated protection against experimental pneumococcal sepsis and suggest that the efficacy of antibody-mediated protection depends on immunomodulation. Taken together, our data extend the association between antibody-mediated protection and immunomodulation to protection against systemic pneumococcal infection and to a clinically important serotype often responsible for pneumococcal sepsis.

1α,25-Dihydroxyvitamin D3 modulates the murine antibody response to pneumococcal capsular polysaccharide serotype 3 through IL-12

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is a steroid hormone that regulates calcium metabolism. Besides, 1alpha,25(OH)(2)D(3 )also has pronounced immunomodulatory effects: it strongly inhibits dendritic cell (DC) maturation and impairs IL-12 production. We studied the effect of 1alpha,25(OH)(2)D(3 )on the antibody response to pneumococcal capsular polysaccharide (caps-PS) serotype 3. 1alpha,25(OH)(2)D(3) inhibited the IgG2a antibody response to caps-PS serotype 3. Besides, 1alpha,25(OH)(2)D(3) also inhibited IL-12 production and maturation of DC. Anti-IL-12 and exogenous IL-12, respectively, inhibited and stimulated the IgG2a antibody response to caps-PS serotype 3. Exogenous IL-12 abrogated the effect of 1alpha,25(OH)(2)D(3) on the IgG2a antibody response to caps-PS serotype 3, indicating that the effect of 1alpha,25(OH)(2)D(3) on the IgG2a antibody response to caps-PS serotype 3 was mediated through IL-12. In conclusion, we demonstrate that 1alpha,25(OH)(2)D(3) has an inhibitory effect on the IgG2a antibody response to caps-PS serotype 3, and that this effect was mediated trough IL-12.

Avidity, potency, and cross-reactivity of monoclonal antibodies to pneumococcal capsular polysaccharide serotype 6B.

Many pneumococcal capsular polysaccharides (PSs) are similar in structure, and a pneumococcal antibody often binds to all of the PSs with a similar structure. Yet, these cross-reactive antibodies may bind to the structurally related pneumococcal capsular PSs with an avidity too low to be effective. If memory B cells producing such weakly cross-reactive antibodies are elicited with pneumococcal conjugate vaccines, the memory cells for low-avidity antibodies could compromise the subsequent immune responses to the cross-reactive PS (original antigenic sin). To investigate these issues, we produced 14 hybridomas secreting monoclonal antibodies (MAbs) to the capsular PS of Streptococcus pneumoniae serotype 6B by immunizing BALB/c mice with antigens containing 6B PS and studied their epitope, avidity, in vitro opsonizing capacity, in vivo protective capacity, and "antigen binding titer" by enzyme-linked immunosorbent assay (ELISA) of 6A and 6B capsular PSs. Six MAbs bound to the non-cross-reactive 6B-specific epitope, and seven MAbs bound to the cross-reactive epitope present in both 6A and 6B PSs One MAb (Hyp6BM6) revealed a novel epitope. This epitope was found on 6A PS in solution, but not on 6A PS adsorbed onto the plastic surface of the ELISA plates. The avidity of the MAb for 6A or 6B PS ranged from 7.8 x 10(6) M(-1) to 4.1 x 10(11) M(-1). No MAbs were weakly cross-reactive, since none of the cross-reactive MAbs showed any tendency toward having less avidity to 6A PS (the cross-reactive PS) than to 6B PS. Avidity influenced the results of several antibody assays. When all of the hybridomas were examined, avidity strongly correlated with the titer of a unit amount of MAb to bind antigen-coated ELISA plates (r = 0.91) or to opsonize pneumococci in vitro (r = -0.85). Because both assay results are avidity dependent, the ELISA and the opsonization assay results were strongly correlated (r = 0.91), regardless of avidity. Avidity also correlated with the potency of a MAb to passively protect mice against pneumococcal infections. When only the immunoglobulin G hybridomas were examined, little increase in opsonizing capacity and in vivo protective potency was observed above 10(9) M(-1). Taken together, an ELISA measuring antigen binding titer may be an adequate measure of the protective immunity induced with pneumococcal vaccines, and the absence of a partially cross-reactive MAb suggests that antigenic sin may not be significant in responses to vaccines against the S. pneumoniae 6B serotype.

Characterization of pneumococcal specific antibodies in healthy unvaccinated adults

Unlike the elderly, healthy middle aged adults are at relatively low risk of acquiring serious pneumococcal disease. An explanation that has been proposed is that people in this age group have significant amounts of serum antibody (primarily IgG2) that react with many pneumococcal capsular polysaccharide serotypes. The level of antibody can be as high as several hundred micrograms per milliliter of blood for some serotypes. A significant component of this reactivity is directed toward the conserved C-polysaccharide depletion. Even after C-polysaccharide depletion, which is included as a routine part of the assay to determine antibody levels, resting antibody levels in a normal healthy adult population can vary widely. We have analyzed the reactivity of serum from 76 people to 16 pneumococcal capsular polysaccharide serotypes. The antibody reactivities to 13 of 16 serotypes are highly correlated with one another. Depletion of serum with C-polysaccharide and purified capsular polysaccharide inhibited antibody binding to type specific capsular polysaccharide. Cross-serotype inhibition of antibody binding was also observed. This indicates that there are materials contained within the pneumococcal polysaccharides that contribute to the cross-reactivity of serum antibodies in people that have not been vaccinated with the pneumococcal vaccine.

Automated electrokinetic analysis; description and application in virology and cell biology

The electrophoretic mobility (EPM) of selected macromolecules in solution was shown to be accurately determined using an automated electrokinetic analyzer, the PenKem S3000. In addition, the S3000 was used to monitor the effects of T4 phage infection on the EPM of Escherichia coli B. EPM, expressed as the ratio of velocity in μm/sec to field strength in V/cm, was measured for calf thymus DNA, for pneumococcal capsular polysaccharide serotype 3 (PCP-3), and for bovine serum albumin (BSA) unbound in solution; values of −3.05, −2.736 and −1.176, respectively, were obtained. The EPM of these macromolecules remained the same when they were bound to latex beads. The S3000 may therefore be suitable for measurement of the EPM of unbound macromolecules. The EPM of T4 phage in solution was measured to be −1.203. However, both the zwitterionic latex-bound T4 phage as well as T4 phage disrupted by ultrasonication exhibited an EPM of approximately −2.50, suggesting to us that binding to zwitterionic latex may cause release of phage DNA. The notion that phage DNA is responsible for the increased negative charge was supported by the observation that the EPM of E. coli B increased to the level of free DNA within 5 min when E. coli B (the host cell for phage T4) had been exposed to 10 phage particles per cell. Electronmicrographs of phage infected E. coli B cells showed numerous strands of free DNA at the bacterial surface. It is concluded that the S3000 not only measures the EPM of macromolecules in solution but that the instrument can be used also to monitor the behavior of the host cell surface in response to attachment of viral particles.