Low invasiveness of pneumococcal serotype 11A is linked to ficolin-2 recognition of O-acetylated capsule epitopes and lectin complement pathway activation.

BACKGROUND: The divergent epidemiological behavior of Streptococcus pneumoniae serotypes suggests that serotype-specific features such as capsule O-acetylation influence the propensity of a strain to cause invasive pneumococcal disease (IPD). We hypothesize that innate host factors mediate the observed negative association between IPD and the serotype 11A (ST11A) capsule O-acetyltransferase gene, wcjE. METHODS: We evaluated the ability of ficolin-2, an initiator of the lectin complement pathway that was previously shown to bind ST11A pneumococci, to recognize and mediate complement-dependent opsonophagocytosis of different pneumococcal serotypes. We supplemented findings with an epidemiological meta-analysis comparing invasiveness of the 30 most prevalent pneumococcal serotypes. RESULTS: Ficolin-2 bound ST11A capsule polysaccharide and other wcjE-containing pneumococcal serotypes, except ST9V and ST20B. Ficolin-2 did not bind wcjE-null serotypes, including the wcjE-null variant of ST11A, ST11E. We observed C1q-independent complement deposition and phagocytic killing of pneumococci expressing ST11A but not those expressing ST11E. Inhibition of ficolin-2 binding abrogated ST11A-associated complement deposition and phagocytosis. In children, invasiveness of ST11A was the lowest among serotypes tested in our meta-analysis, while ST9V was among the highest. CONCLUSIONS: Ficolin-2 mediates serum protection by recognizing specific O-acetylated epitopes of pneumococcal capsule polysaccharides, exemplifying a novel host-microbe interaction that innately offers serotype-specific immunity to IPD.

Ficolin-2 Lectin Complement Pathway Mediates Capsule-Specific Innate Immunity Against Invasive Pneumococcal Disease.

Reports conflict regarding which lectin-microbial ligand interactions elicit a protective response from the lectin pathway (LP) of complement. Using fluorescent microscopy, we demonstrate the human lectin ficolin-2 binds to Streptococcus pneumoniae serotype 11A capsule polysaccharide dependent on the O-acetyltransferase gene wcjE. This triggers complement deposition and promotes opsonophagocytosis of encapsulated pneumococci. Even partial loss of ficolin-2 ligand expression through wcjE mutation abrogated bacterial killing. Ficolin-2 did not interact with any pneumococcal non-capsule structures, including teichoic acid. We describe multiple 11A clonal derivatives expressing varying degrees of wcjE-dependent epitopes co-isolated from single blood specimens, likely representing microevolutionary shifts towards wcjE-deficient populations during invasive pneumococcal disease (IPD). We find epidemiological evidence of wcjE impairing pneumococcal invasiveness, supporting that the LP's ficolin-2 axis provides innate, serotype-specific serological protection against IPD. The fact that the LP is triggered by only a few discrete carbohydrate ligands emphasizes the need to reevaluate its impact in a glycopolymer-specific manner.

Potentiating pneumococcal glycoconjugate vaccine PCV13 with saponin adjuvant VSA-1.

VSA-1 is a semisynthetic saponin adjuvant prepared from naturally occurring Momordica saponin and capable of stimulating antigen-specific humoral and cellular immune responses. Its immunostimulating activity in enhancing the immune responses induced by the clinical glycoconjugate pneumococcal vaccine PCV13 is compared with QS-21 in female BALB/c mice. Both VSA-1 and QS-21 boosted IgG and opsonic antibodies titers against seven selected serotypes, including serotypes 3, 14, and 19A that are involved in most PCV13 breakthroughs. Since VSA-1 is much more accessible and of lower toxicity than QS-21, it can be a practical saponin immunostimulant to be included in a new glycoconjugate pneumococcal vaccine formulation.

Pneumococcal serotypes causing pneumonia with pleural effusion in pediatric patients.

To determine the prevalence of serotypes of Streptococcus pneumoniae responsible for pneumonia with pleural effusion, we determined the capsular polysaccharide (PS) type directly on 49 pleural fluid specimens collected from pediatric patients during 2007 to 2009 with laboratory-confirmed pneumococcal pneumonia by using monoclonal antibodies and a multiplex, bead array immunoassay. Because the fluids had to be heated to remove nonspecific reactivity before being tested in the immunoassay and type 19A PS is heat labile, the pleural fluid samples were also tested for serotype 19A capsule gene locus by PCR. Use of the multiplex immunoassay combined with type-specific 19A PCR allowed for serotype determination on 40 of 49 pleural fluids. Pneumococcal pneumonia with pleural effusion was associated with a limited number of serotypes, with types 1, 3, 7F/A, and 19A accounting for 75% of the typeable cases. The concentration of capsular PS in the pleural fluids was often greater than 1 µg/ml and sufficient to inhibit the opsonic capacity of sera from individuals who had received the 23-valent pneumococcal PS vaccine. Based on the serotypes observed before and after introduction of the 7-valent pneumococcal conjugate vaccine, the recently licensed 13-valent pneumococcal conjugate vaccine may reduce the incidence of pneumonia with pleural effusions.

Nasopharyngeal pneumococcal carriage of children attending day care centers in Korea: comparison between children immunized with 7-valent pneumococcal conjugate vaccine and non-immunized.

To confirm the effect of 7-valent pneumococcal conjugate vaccine (PCV7), pneumococcal nasopharyngeal (NP) carriage was compared between vaccinated (3 + 1 doses PCV7) and non-vaccinated children. Vaccinated subjects were recruited from highly vaccinated regions (≥ 60%), Seoul and Incheon whereas control subjects were recruited from Jeju Island where vaccination rates are low (< 15%). NP swabs were obtained from 400 children aged 18-59 months. Serotype and antibiotic susceptibility was analyzed. Pneumococcal carriage rate was 18.0% (36/200) and 31.5% (63/200) for the vaccinated and control group, respectively. Among those vaccinated, 41.7% (15/36) of the serotypes were vaccine-related type (VRT: 6A, 6C, 19A) with the most common serotype 6C. The next common type was non-typable/non-capsule 30.6% (11/36) followed by non-vaccine type 16.7% (6/36) and vaccine type (VT) serotypes were found in only 11.1% (4/36). In contrast, 52.4% (33/63) of the isolates in the control group were VT. Resistance rates for penicillin and erythromycin were lower in the vaccine group (vaccine vs control; penicillin 45.2% vs 71.4%, erythromycin 74.2% vs 90.5%, P < 0.05). Multi-drug resistance was also lower in vaccinated subjects (vaccine vs control; 45.2% vs 69.8%, P < 0.05). PCV7 reduces carriage in VT which leads to replacement of pneumococci by antibiotic susceptible VRT or non-vaccine type strains.

A Common Food Glycan, Pectin, Shares an Antigen with Streptococcus pneumoniae Capsule.

We are exposed daily to many glycans from bacteria and food plants. Bacterial glycans are generally antigenic and elicit antibody responses. It is unclear if food glycans' sharing of antigens with bacterial glycans influences our immune responses to bacteria. We studied 14 different plant foods for cross-reactivity with monoclonal antibodies (MAbs) against 24 pneumococcal serotypes which commonly cause infections and are included in pneumococcal vaccines. Serotype 15B-specific MAb cross-reacts with fruit peels, and serotype 10A MAb cross-reacts with many natural and processed plant foods. The serotype 10A cross-reactive epitope is terminal 1,6-linked ß-galactose [ßGal(1-6)], present in the rhamno-galacturonan I (RG-I) domain of pectin. Despite wide consumption of pectin, the immune response to 10A is comparable to the responses to other serotypes. An antipectin antibody can opsonize serotype 10A pneumococci, and the shared ßGal(1-6) may be useful as a simple vaccine against 10A. Impact of food glycans should be considered in host-pathogen interactions and future vaccine designs.IMPORTANCE The impact of food consumption on vaccine responses is unknown. Streptococcus pneumoniae (the pneumococcus) is an important human pathogen, and its polysaccharide capsule is used as a vaccine. We show that capsule type 10A in a pneumococcal vaccine shares an antigenic epitope, ßGal(1-6), with pectin, which is in many plant foods and is widely consumed. Immune response to 10A is comparable to that seen with other capsule types, and pectin ingestion may have little impact on vaccine responses. However, antibody to pectin can kill serotype 10A pneumococci and this shared epitope may be considered in pneumococcal vaccine designs.

A High-throughput Shigella-specific Bactericidal Assay.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

Development, Interlaboratory Evaluations, and Application of a Simple, High-Throughput Shigella Serum Bactericidal Assay.

Shigella is an important cause of diarrhea worldwide, with serotypes Shigella flexneri 2a, S. flexneri 3a, and Shigella sonnei demonstrating epidemiological prevalence. Many development efforts are focused on Shigella lipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate vaccines are immunogenic and efficacious. Immunization with Shigella vaccines containing LPS can elicit antibodies capable of killing Shigella in a serotype-specific manner. Thus, to facilitate Shigella vaccine development, we have developed a serum bactericidal assay (SBA) specific for three Shigella serotypes that measures killing of target bacteria at multiple serum dilutions and in the presence of exogenous complement. The SBA has a high analytical throughput and uses simple technologies and readily available reagents. The SBA was characterized with human sera with bactericidal antibodies against S. flexneri 2a, S. flexneri 3a, and S. sonnei Purified LPS of a homologous serotype, but not a heterologous serotype, inhibited bacterial killing. Assessment of precision found median intra-assay precision to be 13.3% and median interassay precision to be 19 to 30% for the three serotypes. The SBA is linear, with slight deviations for samples with low (~40) killing indices. The SBA was sensitive enough to allow about 100-fold predilution of serum samples. Repeat assays yielded results with less than 2-fold deviations, indicating the robustness of the assay. Assay results from four different laboratories were highly comparable when normalized with a reference serum. The Shigella SBA, combined with a reference serum, should facilitate the development of Shigella vaccines across the field.IMPORTANCEShigella is an important cause of diarrhea worldwide, and efforts are ongoing to produce a safe and effective Shigella vaccine. Although a clear immune correlate of protection has not been established, antibodies with bactericidal capacity may provide one means of protecting against shigellosis. Thus, it is important to measure the functional capacity of antibodies, as opposed to only binding activity. This article describes a simple, robust, and high-throughput serum bactericidal assay capable of measuring Shigella-specific functional antibodies in vitro We show for the first time that this assay was successfully performed by multiple laboratories and generated highly comparable results, particularly when SBA titers were normalized using a reference standard. The serum bactericidal assay, along with a reference serum, should greatly facilitate Shigella vaccine development.

Genetic, biochemical, and serological characterization of a new pneumococcal serotype, 6H, and generation of a pneumococcal strain producing three different capsular repeat units.

Streptococcus pneumoniae clinical isolates were recently described that produced capsular polysaccharide with properties of both serotypes 6A and 6B. Their hybrid serological property correlated with mutations affecting the glycosyltransferase WciP, which links rhamnose to ribitol by an α(1-3) linkage for serotypes 6A and 6C and an α(1-4) linkage for serotypes 6B and 6D. The isolates had mutations in the triad residues of WciP that have been correlated with enzyme specificity. The canonical triad residues of WciP are Ala192-Ser195-Arg254 for serotypes 6A and 6C and Ser192-Asn195-Gly254 for serotypes 6B and 6D. To prove that the mutations in the triad residues are responsible for the hybrid serotype, we introduced the previously described Ala192-Cys195-Arg254 triad into a 6A strain and found that the change made WciP bispecific, resulting in 6A and 6B repeat unit expression, although 6B repeat unit production was favored over production of 6A repeat units. Likewise, this triad permitted a 6C strain to express 6C and 6D repeat units. With reported bispecificity in WciN, which adds either glucose or galactose as the second sugar in the serogroup 6 repeat unit, the possibility exists for a strain to simultaneously produce all four serogroup 6 repeat units; however, when genes encoding both bispecific enzymes were introduced into a 6A strain, only 6A, 6B, and 6D repeat units were detected serologically. Nonetheless, this may be the first example of a bacterial polysaccharide with three different repeat units. This strategy of expressing multiple repeat units in a single polymer is a novel approach to broadening vaccine coverage by eliminating the need for multiple polysaccharide sources to cover multiple serogroup members.

Streptococcus pneumoniae serotype 6C presenting as recurrent prosthetic knee joint infection in a patient with a history of congenital asplenia and underlying autoimmune disease: a case report and literature review.

This report describes a case of primary Streptococcus pneumoniae bacteremia with prosthetic joint infection caused by serotype 6C with recurrent infection in a patient with a history of congenital asplenia and underlying autoimmune disease. Isolates from the primary and recurrent infections were determined to be indistinguishable by pulsed-field gel electrophoresis. This study expands the conditions associated with recurrent invasive pneumococcal disease caused by serotype 6C.

Development of an automated and multiplexed serotyping assay for Streptococcus pneumoniae.

Streptococcus pneumoniae expresses more than 90 capsule types, and currently available pneumococcal vaccines are designed to provide serotype-specific protection. Consequently, serotyping of pneumococcal isolates is important for determining the serotypes to be included in pneumococcal vaccines and to monitor their efficacy. Yet serotyping of pneumococcal isolates has remained a significant technical challenge. By multiplexing many assays, we have now developed a simple yet comprehensive serotyping assay system that can not only identify all known pneumococcal serotypes but also subdivide nontypeable (NT) isolates into those with or without the conventional capsule locus. We have developed this assay system to require only six key reagents: two are used in one multiplex inhibition-type immunoassay, and four are required in two multiplex PCR-based assays. The assay system is largely automated by a seamless combination of monoclonal antibody-based and PCR-based multiplex assays using the flow cytometric bead array technology from Luminex. The assay system has been validated with a panel of pneumococci expressing all known pneumococcal serotypes and was found to be easily transferable to another laboratory.

Type distribution of serogroup 6 Streptococcus pneumoniae and molecular epidemiology of newly identified serotypes 6C and 6D in China.

The recently determined serotypes 6C and 6D Streptococcus pneumoniae, as well as subtypes 6B-I and 6B-II, were not reported in China. Among the 171 invasive isolates, 19 were identified as serogroup 6. There were equal distribution (42.1%) of 6B-I and 6B-II, 15.8% of 6A and lack of 6C and 6D. Among 1662 noninvasive isolates, 210 were identified as serogroup 6. The rates of types 6A, 6B-I, 6B-II, 6C, and 6D were 42.4%, 21.0%, 29.1%, 4.8%, and 2.9%, respectively. Subtype 6B-II was more resistant to antibiotics than others. The main sequence types (STs) of serotype 6C and 6D isolates were ST2912 and ST982, respectively. These results suggested that all recognized types of serogroup 6 can be found in China and that subtype 6B-II was more drug resistant. The epidemic STs of serotype 6C and 6D did not show genetic association with the STs spreading in other countries.

Structure of the capsular polysaccharide of pneumococcal serotype 11A reveals a novel acetylglycerol that is the structural basis for 11A subtypes.

We have undertaken a structural assessment of Streptococcus pneumoniae 11A polysaccharide as well as two clinical isolates related to 11A. The clinical isolates were labeled 11Aalpha and 11Abeta. The result of our experiments is a revision to the old structure for S. pneumoniae 11A polysaccharide. The new structure differs from the old structure in both the primary connectivities and acetylation pattern. We also show that 11A contains an acetylglycerol-PO4 moiety, a substitution that is heretofore unknown in the bacterial polysaccharide literature. The two clinical isolates were also structurally characterized. 11Aalpha was determined to be identical to 11A. 11Abeta is a new serotype, which differs from 11A in the absence of the acetylation of the glycerol-PO4 moiety and a different acetylation pattern of the saccharides. Thus, we propose that the acetylglycerol is the structural basis for 11Aalpha and 11Abeta subtypes.

Identification of 3-O-acetylglycerol, a novel structural element in bacterial polysaccharides.

We have discovered a novel bacterial polysaccharide structural element, 3-O-acetylglycerol, in the Streptococcus pneumoniae ST11A polysaccharide: This moiety was elucidated through a combination of homonuclear and heteronuclear 1D and 2D NMR experiments using (1)H, (13)C, and (31)P in various combinations. The 3-O-acetylglycerol moiety is substoichiometrically O-acetylated in ST11A; yet, key connectivities that unequivocally show O-acetylation at the glycerol are provided by the long-range correlations from the acetate methyl groups to the glycerol in the (1)H-(13)C HMBC spectrum. Additionally, we clarify the (1)H-(31)P assignments previously presented.

Rapid multiplex assay for serotyping pneumococci with monoclonal and polyclonal antibodies.

We have developed and characterized a rapid semiautomated pneumococcal serotyping system incorporating a pneumococcal lysate preparation protocol and a multiplex serotyping assay. The lysate preparation incorporates a bile solubility test to confirm pneumococcal identification that also enhances assay specificity. The multiplex serotyping assay consists of 24 assays specific for 36 serotypes: 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/(15C), 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F. The multiplex assay requires a flow cytometer, two sets of latex particles coated with pneumococcal polysaccharides, and serotype-specific antibodies. Fourteen newly developed monoclonal antibodies specific for common serotypes and a pool of polyclonal rabbit sera for some of the less-common serotypes are used. The two monoclonal antibodies specific for serotypes 18C and 23F recognize serotype-specific epitopes that have not been previously described. These monoclonal antibodies make the identification of the 14 common serotypes invariant. The specificity of the serotyping assay is fully characterized with pneumococci of all known (i.e., 90) serotypes. The assay is sensitive enough to use bacterial lysates diluted 20 fold. Our serotyping system can identify not only all the serotypes in pneumococcal vaccines but also most (>90%) of clinical isolates. This system should be very useful in serotyping clinical isolates for evaluating pneumococcal vaccine efficacy.

Efficiency of a pneumococcal opsonophagocytic killing assay improved by multiplexing and by coloring colonies.

For evaluating pneumococcal vaccines, the opsonophagocytic killing assay (OPKA) is useful as a supplement to the pneumococcal antibody enzyme-linked immunosorbent assay (ELISA). However, evaluations of pneumococcal vaccines require the determination of antibody responses to 7 to 11 serotypes, and the OPKA is tedious to perform and requires more serum than the ELISA. Consequently, the OPKA is infrequently used for evaluating pneumococcal vaccines. To overcome these limitations, we have developed a simple multiplexed (double-serotype) OPKA by using antibiotic-resistant pneumococci for nine serotypes. Serotype 6B, 9V, 19A, and 23F strains were made streptomycin resistant, and serotype 4, 6A, 14, 18C, and 19F strains were made optochin resistant. The multiplexed OPKA was the same as the single-serotype OPKA except for two changes. First, the target bacteria were a mixture of one streptomycin-resistant strain and one optochin-resistant strain. Second, the surviving bacteria of each serotype were enumerated by plating on Todd-Hewitt agar plates with yeast extract and an agar overlay containing the appropriate antibiotics and 2,3,5-triphenyl tetrazolium chloride. The performance of the multiplexed OPKA was evaluated by analyzing 28 serum samples from adults immunized with a 23-valent polysaccharide vaccine by using the single-serotype OPKA and the multiplexed OPKA. The multiplexed OPKA was specific for the desired serotypes. The multiplexed and conventional OPKAs had comparable assay sensitivities and produced results that were highly correlated (r(2) values ranging from 0.92 to 0.98) for all nine serotypes. A simple modification of the conventional OPKA produces a multiplexed assay that greatly reduces effort, reagents, and the necessary amount of serum.

Peptide mimotopes of pneumococcal capsular polysaccharide of 6B serotype: a peptide mimotope can bind to two unrelated antibodies.

Two groups of bacteriophage clones displaying the antigenic properties of serotype 6B pneumococcal capsular polysaccharide (PS) were obtained from different phage libraries expressing random heptameric peptides. One group, biopanned with a mouse mAb (Hyp6BM1), is comprised of 17 phage clones expressing 10 unique sequences of linear peptides. The other group, selected with another mAb (Hyp6BM8), contained six clones, all of which expressed the identical circular peptide. Phage clones expressing the linear peptides (e.g., PhaM1L3) bound only to Hyp6BM1, but not other 6B PS-specific mAb, and their binding could be inhibited with pneumococcal capsular type 6B PS only. In contrast, a phage clone expressing the circular peptide (PhaM8C1) cross-reacted with several other 6B PS-specific mAbs, and their binding could be inhibited with pneumococcal capsular PS of 6A and 6B serotypes. Two short peptides, PepM1L3 and PepM8C1, reflecting the peptide inserts of the corresponding phage clones, could inhibit the binding of the two clones to their respective mAb. Interestingly, the peptide insert in PhaM8C1 was identical to that in PhaB3C4, a previously reported mimotope of alpha(2-->8) polysialic acid, Neisseria meningitidis group B PS. Indeed, PhaM8C1 bound to HmenB3 (a meningococcal Ab), and their association could be inhibited with alpha(2-8) polysialic acid, but not with 6B PS. Conversely, alpha(2-8) polysialic acid could not inhibit the binding of PhaM8C1 to Hyp6BM8. The two-dimensional nuclear magnetic resonance studies indicate that PepM8C1 peptide can assume several conformations in solution. The ability of this peptide to assume multiple conformations might account for its ability to mimic more than one Ag type.

Immunogenic protein contaminants in pneumococcal vaccines.

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.