Base hydrolysis of phosphodiester bonds in pneumococcal polysaccharides

A comprehensive study of the base hydrolysis of all phosphodiester bond-containing capsular polysaccharides of the 23-valent pneumococcal vaccine is described here. Capsular polysaccharides from serotypes 6B, 10A, 17F, 19A, 19F, and 20 contain a phosphodiester bond that connects the repeating units in these polysaccharides (also referred to as backbone phosphodiester bonds), and polysaccharides from serotypes 11A, 15B, 18C, and 23F contain a phosphodiester bond that links a side chain to their repeating units. Molecular weight measurements of the polysaccharides, using high performance size exclusion chromatography with tandem multiangle laser light scattering and refractive index detection, was used to evaluate the kinetics of hydrolysis. The measurement of molecular weight provides a high degree of sensitivity in the case of small extents of reaction, thus allowing reliable measurements of the kinetics over short times. Pseudo-first-order rate constants for these polysaccharides were estimated using a simple model that accounts for the polydispersity of the starting sample. It was found that the relative order of backbone phosphodiester bond instability due to base hydrolysis was 19A > 10A > 19F > 6B > 17F, 20. Degradation of side-chain phosphodiester bonds was not observed, although the high degree of sensitivity in measurements is lost in this case, due to the low contribution of the side chains to the total polysaccharide molecular weight. In comparison with literature data on pneumococcal polysaccharide 6A, 19A was found to be the more labile, and hence appears to be the most labile pneumococcal polysaccharide studied to date. The rate of hydrolysis increased at higher pH and in the presence of divalent cation, but the extent was lower than expected based on similar data on RNA. Finally, the differences in the phosphodiester bond stabilities were analyzed by considering stereochemical factors in these polysaccharides. These results also provide a framework for evaluation of molecular integrity of phosphodiester-bond-containing polysaccharides in different solution conditions.     

The quantitative immunochemical determination of pneumococcal and meningococcal capsular polysaccharides by light scattering rate nephelometry

A quantitative nephelometric method was used for the measurement of the individual pneumococcal, as well as meningococcal, polysaccharides in the polyvalent vaccine final containers. This method is simple, rapid, inexpensive, and provides both qualitative and quantitative analyses of the polyvalent polysaccharide vaccines. By this method the individual pneumococcal types, 1, 2, 3, 4, 6A, 7F, 8, 9N, 12F, 14, 18C, 19F, 23F and 25 polysaccharides, were found to be present at 90-114% of the manufacturer's indicated concentrations; meningococcal group A, C, Y and W135 polysaccharides were at 90-108% of the manufacturer's listed concentrations. This nephelometric method coupled with gel filtration can also be used for measurement of the molecular sizes or stability of individual polysaccharides in the final container. Pneumococcal polysaccharide types 3, 6A, 9N and 19F, used as representative types, were treated with 0.5 N hydrochloric acid. The molecular sizes for types 3 and 9 N polysaccharides were stable to acid treatment. In contrast, types 6A and 19F polysaccharides were degraded. Heating meningococcal groups A, C, Y and W135 polysaccharides at 37 degrees C for 48 h did not affect their molecular size in the polyvalent vaccine.     

Type-specific enzyme immunoassay for detection of pneumococcal capsular polysaccharide antigens in nasopharyngeal specimens

We developed a new competitive EIA method for the demonstration of pneumococcal capsular polysaccharides from respiratory samples. The pediatric types 4, 6B, 9V, 14, 18C, 19F and 23F were selected for this study, because these capsular polysaccharides were included in the first heptavalent pneumococcal conjugate vaccines, which were used in the Finnish Otitis Media Vaccine Trial. Sensitivity of the EIA tests for purified polysaccharide antigens varied between 5 and 100 ng/ml, depending on the type. The assays performed well in 100 nasopharyngeal samples (NPS) samples processed through an enrichment culture, with an almost 100% sensitivity compared with routine culture. The method appeared type-specific, except that EIA for 6B capsule also detected 6A. The method is applicable for type-specific identification of pneumococcus in carriage studies.     

Sequence diversity within the capsular genes of Streptococcus pneumoniae serogroup 6 and 19

The main virulence factor of Streptococcus pneumoniae is the capsule. The polysaccharides comprising this capsule are encoded by approximately 15 genes and differences in these genes result in different serotypes. The aim of this study was to investigate the sequence diversity of the capsular genes of serotypes 6A, 6B, 6C, 19A and 19F and to explore a possible effect of vaccination on variation and distribution of these serotypes in the Netherlands. The complete capsular gene locus was sequenced for 25 serogroup 6 and for 20 serogroup 19 isolates. If one or more genes varied in 10 or more base pairs from the reference sequence, it was designated as a capsular subtype. Allele-specific PCRs and specific gene sequencing of highly variable capsular genes were performed on 184 serogroup 6 and 195 serogroup 19 isolates to identify capsular subtypes. This revealed the presence of 6, 3 and a single capsular subtype within serotypes 6A, 6B and 6C, respectively. The serotype 19A and 19F isolates comprised 3 and 4 capsular subtypes, respectively. For serogroup 6, the genetic background, as determined by multi locus sequence typing (MLST) and multiple-locus variable number of tandem repeat analysis (MLVA), seemed to be closely related to the capsular subtypes, but this was less pronounced for serogroup 19 isolates. The data also suggest shifts in the occurrence of capsular subtypes within serotype 6A and 19A after introduction of the 7-valent pneumococcal vaccine. The shifts within these non-vaccine serotypes might indicate that these capsular subtypes are filling the niche of the vaccine serotypes. In conclusion, there is considerable DNA sequence variation of the capsular genes within pneumococcal serogroup 6 and 19. Such changes may result in altered polysaccharides or in strains that produce more capsular polysaccharides. Consequently, these altered capsules may be less sensitive for vaccine induced immunity.     

Novel vaccine strategies with protein antigens of Streptococcus pneumoniae

Infections caused by Streptococcus pneumoniae (pneumococcus) are a major cause of mortality throughout the world. This organism is primarily a commensal in the upper respiratory tract of humans, but can cause pneumonia in high-risk persons and disseminate from the lungs by invasion of the bloodstream. Currently, prevention of pneumococcal infections is by immunization with vaccines which contain capsular polysaccharides from the most common serotypes causing invasive disease. However, there are more than 90 antigenically distinct serotypes and there is concern that serotypes not included in the vaccines may become more prevalent in the face of continued use of polysaccharide vaccines. Also, certain high-risk groups have poor immunological responses to some of the polysaccharides in the vaccine formulations. Protein antigens that are conserved across all capsular serotypes would induce more effective and durable humoral immune responses and could potentially protect against all clinically relevant pneumococcal capsular types. This review provides a summary of work on pneumococcal proteins that are being investigated as components for future generations of improved pneumococcal vaccines.     

Effect of vaccination against pneumococcal infection in children with type 1 diabetes mellitus

Vaccination with polysaccharide pneumococcal vaccine "Pneumo 23" (Sanofi Pasteur, France) was performed in 31 children with type 1 diabetes mellitus (DM1) as well as in 19 children with respiratory tract diseases (asthma, chronic pneumonia), which formed comparison group. Fourty-three unvaccinated children with DM1 were included in the control group. Dynamics of IgG levels to mixture of pneumococcal polysaccharides (PS) included in the vaccine as well as to PS of serotypes 3, 6B, 9N, 23F, and to cell wall polysaccharides of Streptococcus pneumoniae were assessed. Using ELISA method, significant increase of IgG levels to mixture of PS and to PS of pneumococcal serotype 3 was detected. Although intensity of immune response to vaccination in children with respiratory diseases was significantly higher compared to children with DM1 (mean geometric titer of antibodies, proportion of patients with high antibody titers, and with 4-fold seroconversion). Development of methods to strengthen immune response in children with DM1 vaccinated against pneumococcal infection is required.     

An immunoenzyme system for determining antibodies to Streptococcus pneumoniae polysaccharides in biological fluids

The enzyme immunoassay (EIA) system for the determination of antibodies to capsular polysaccharides of pneumococci, serotypes 1, 3, 6B, 8, 9N, 15F, 23F, and C-polysaccharide has been developed on the basis of poly-L-lysin-modified antigens. The use of isotype-specific conjugates in this system permits the detection of IgG and IgA antibodies in different biological fluids: blood serum, pleural fluid, saliva, milk. Samples obtained from children with pneumococcal infection and from nursing mothers have been studied. As shown in this study, the EIA system can be used for the evaluation of the dynamics of pneumococcal infection in children.     

Control of components of bacterial polysaccharide vaccines by physical methods

Analysis of polysaccharide components of meningococcal- and pneumococcal vaccines was carried out by proton n.m.r. spectroscopy and gas chromatography. The meningococcal polysaccharides were of high purity but showed differences in degree and position of O-acetylation between manufacturers. The level of contamination of the pneumococcal polysaccharides by C-substance was quantified. These methods provide an alternative to immunological methods for determining serotype and purity.     

Pneumococcal surface protein A and new approaches for pneumococcal vaccine development

The problem of pneumococcal infections is pressing for the whole world. Existing vaccines based only on pneumococci polysaccharide antigens or polysaccharide antigens and diphtherial anatoxin are not capable of protecting from all serotypes of the microorganism. Reasonability of creation of pneumococcal vaccine based on surface proteins of Streptococcus pneumoniae is discussed in the literature. One of such key pneumococcal proteins is pneumococcal surface protein A (PSPA), because it is detected in all the S. pneumoniae strains, has cross activity and switches B-cell immune response to T-cell. Currently the development of conjugated vaccine based on surface proteins and capsule polysaccharides of pneumococcus seems promising.     

5型肺炎链球菌荚膜多糖结合疫苗的不同糖蛋白比与免疫原性的关系

实验中比较了以不同糖蛋白比结合的5型肺炎链球菌荚膜多糖结合疫苗的免疫原性。以氨还原法制备的不同糖蛋白比的5型多糖结合疫苗分别经腹腔注射NIH小鼠,共免疫3次,间隔2周。末次免疫2周后采血分离血清,以间接ELISA测定血清抗体,t检验统计分析数据。结果显示,生理盐水组(R组)无免疫原性;5型荚膜多糖组(H组)免疫原性低,无免疫加强效应;结合疫苗M组、N组第1、2针有免疫加强效应,第2、3针无免疫加强效应,而F组和Q组的两针次间均有明显的免疫加强效应。说明5型肺炎链球菌荚膜多糖结合疫苗的糖与蛋白比较高时能获得较好的免疫原性。     

Strategy of glycerolipid separation and quantitation by complementary analytical techniques : Plenary lecture

Although improved systems for chromatographic resolution continue to be developed there is good reason to believe that no single method will be capable of complete separation of all lipid mixtures including the geometric, positional and stereochemical isomers in each molecular species. Furthermore, the chromatographic systems giving the highest resolution usually yield the least complete recoveries of components and require separate procedures of quantitation. It is therefore necessary to develop appropriate strategies that yield the required resolution as a result of consecutive application of complementary analytical techniques. At the present time, the original combination of thin-layer and gas— liquid chromatography has been joined by the combination of thin-layer and liquid, and liquid and gas—liquid chromatography with both liquid and gas—liquid chromatography being frequently coupled to mass spectrometry with computerized data processing. Internal standardization with hydrogen flame ionization provides a simple quantitative detection for gas chromatography, while mass spectrometry serves a similar purpose in liquid chromatography, although a much more extensive calibration may be required for quantitation. Special advantages for both separation and quantitation of most neutral lipid mixtures are derived from enzymic and chemical modification of the samples prior to chromatography. With imaginative work-up of samples, superior qualitative and quantitative results can frequently be obtained by appropriate combination of chromatographic techniques of limited resolving power.     

Development and clinical evaluation of Prevnar 13, a 13-valent pneumocococcal CRM(197) conjugate vaccine

Pneumococcus is the leading cause of bacterial illness in children worldwide. The development, clinical evaluation, and postlicensure impact of the pneumococcal CRM(197) protein conjugate vaccine, PCV13, (Prevnar 13?) builds upon the excellent safety and substantial effectiveness of PCV7 (Prevnar?) in preventing pneumococcal disease in children. PCV13 adds pneumococcal serotypes 1, 3, 5, 6A, 7F, and 19A to serotypes 4, 6B, 9V, 14, 18C, 19F, 23F in PCV7 to provide comprehensive coverage for over 85% of epidemiologically important pneumococcal serotypes in the United States and throughout the world. PCV13 development required demonstration of immunologic responses to the 13 serotypes contained in the vaccine that were noninferior to the responses elicited by PCV7, and demonstration of a satisfactory safety profile. Studies were also performed to demonstrate compatibility with other childhood vaccines. Now licensed in many countries worldwide, PCV13 shows significant promise for expanded protection against pneumococcal disease in children.     

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

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

High Nasopharyngeal Carriage of Non-Vaccine Serotypes in Western Australian Aboriginal People Following 10 Years of Pneumococcal Conjugate Vaccination

Background

Invasive pneumococcal disease (IPD) continues to occur at high rates among Australian Aboriginal people. The seven-valent pneumococcal conjugate vaccine (7vPCV) was given in a 2-4-6-month schedule from 2001, with a 23-valent pneumococcal polysaccharide vaccine (23vPPV) booster at 18 months, and replaced with 13vPCV in July 2011. Since carriage surveillance can supplement IPD surveillance, we have monitored pneumococcal carriage in western Australia (WA) since 2008 to assess the impact of the 10-year 7vPCV program.

Methods

We collected 1,500 nasopharyngeal specimens from Aboriginal people living in varied regions of WA from August 2008 until June 2011. Specimens were cultured on selective media. Pneumococcal isolates were serotyped by the quellung reaction.

Results

Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis were carried by 71.9%, 63.2% and 63.3% respectively of children <5 years of age, and 34.6%, 22.4% and 27.2% of people ≥5 years. Of 43 pneumococcal serotypes identified, the most common were 19A, 16F and 6C in children <5 years, and 15B, 34 and 22F in older people. 7vPCV serotypes accounted for 14.5% of all serotypeable isolates, 13vPCV for 32.4% and 23vPPV for 49.9%, with little variation across all age groups. Serotypes 1 and 12F were rarely identified, despite causing recent IPD outbreaks in WA. Complete penicillin resistance (MIC ≥2µg/ml) was found in 1.6% of serotype 19A (5.2%), 19F (4.9%) and 16F (3.2%) isolates and reduced penicillin susceptibility (MIC ≥0.125µg/ml) in 24.9% of isolates, particularly 19F (92.7%), 19A (41.3%), 16F (29.0%). Multi-resistance to cotrimoxazole, tetracycline and erythromycin was found in 83.0% of 23F isolates. Among non-serotypeable isolates 76.0% had reduced susceptibility and 4.0% showed complete resistance to penicillin.

Conclusions

Ten years after introduction of 7vPCV for Aboriginal Australian children, 7vPCV serotypes account for a small proportion of carried pneumococci. A large proportion of circulating serotypes are not covered by any currently licensed vaccine.     

Impact of Preceding Flu-Like Illness on the Serotype Distribution of Pneumococcal Pneumonia

Background

Even though the pathogenicity and invasiveness of pneumococcus largely depend on capsular types, the impact of serotypes on post-viral pneumococcal pneumonia is unknown.

Methods and Findings

This study was performed to evaluate the impact of capsular serotypes on the development of pneumococcal pneumonia after preceding respiratory viral infections. Patients with a diagnosis of pneumococcal pneumonia were identified. Pneumonia patients were divided into two groups (post-viral pneumococcal pneumonia versus primary pneumococcal pneumonia), and then their pneumococcal serotypes were compared. Nine hundred and nineteen patients with pneumococcal pneumonia were identified during the study period, including 327 (35.6%) cases with post-viral pneumococcal pneumonia and 592 (64.4%) cases with primary pneumococcal pneumonia. Overall, serotypes 3 and 19A were the most prevalent, followed by serotypes 19F, 6A, and 11A/11E. Although relatively uncommon (33 cases, 3.6%), infrequently colonizing invasive serotypes (4, 5, 7F/7A, 8, 9V/9A, 12F, and 18C) were significantly associated with preceding respiratory viral infections (69.7%, P<0.01). Multivariate analysis revealed several statistically significant risk factors for post-viral pneumococcal pneumonia: immunodeficiency (OR 1.66; 95% CI, 1.10–2.53), chronic lung diseases (OR 1.43; 95% CI, 1.09–1.93) and ICI serotypes (OR 4.66; 95% CI, 2.07–10.47).

Conclusions

Infrequently colonizing invasive serotypes would be more likely to cause pneumococcal pneumonia after preceding respiratory viral illness, particularly in patients with immunodeficiency or chronic lung diseases.     

肺炎球菌表面蛋白A(PspA)及其荚膜多糖交联物的免疫原性和交叉保护作用

分别采用肺炎球菌表面蛋白A(PspA)和PspA-荚膜多糖交联物免疫小鼠,研究PspA及其交联物的免疫特性.用酶联免疫吸附法(ELISA)检测抗原的免疫原性,用动物保护试验检验抗原对肺炎球菌6B,5,1,23F,19F型的交叉免疫效果.实验结果表明:肺炎球菌表面蛋白A及其多糖交联物表现出一定的交叉保护作用,具有较好的应用前景.     

Genetic relatedness of the Streptococcus pneumoniae capsular biosynthetic loci

Streptococcus pneumoniae (the pneumococcus) produces 1 of 91 capsular polysaccharides (CPS) that define the serotype. The cps loci of 88 pneumococcal serotypes whose CPS is synthesized by the Wzy-dependent pathway were compared with each other and with additional streptococcal polysaccharide biosynthetic loci and were clustered according to the proportion of shared homology groups (HGs), weighted for the sequence similarities between the genes encoding the shared HGs. The cps loci of the 88 pneumococcal serotypes were distributed into eight major clusters and 21 subclusters. All serotypes within the same serogroup fell into the same major cluster, but in six cases, serotypes within the same serogroup were in different subclusters and, conversely, nine subclusters included completely different serotypes. The closely related cps loci within a subcluster were compared to the known CPS structures to relate gene content to structure. The Streptococcus oralis and Streptococcus mitis polysaccharide biosynthetic loci clustered within the pneumococcal cps loci and were in a subcluster that also included the cps locus of pneumococcal serotype 21, whereas the Streptococcus agalactiae cps loci formed a single cluster that was not closely related to any of the pneumococcal cps clusters.     

Proteins of Streptococcus pneumoniae: perspectives for development of pneumococcal vaccine

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

Quality control of polyvalent pneumococcal polysaccharide-protein conjugate vaccine by nephelometry.

A nephelometric method was used for quantitative analysis of individual polysaccharides (PSs) in a polyvalent pneumococcal conjugate vaccine using CRM(197) as carrier protein. Using this method, the individual types 4, 6B, 9V, 14, 18C, 19F and 23F PSs were found to range between 82.3 to 119% of the manufacturer's indicated values.During conjugation using reductive amination, pneumococcal PS was first oxidized to introduce aldehyde groups. Higher or lower levels of antigen-antibody reaction were observed in periodate activated and then reduced PS of some serotypes compared to non-treated PS. Use of oxidized and reduced PS may provide an early indication of change in conjugation process. Furthermore, since the final monovalent and polyvalent conjugate vaccines gradually change during the storage period, the nephelometry provides an useful analytical method for stability study of these vaccines.     

Capsular serotype and antibiotic resistance of Streptococcus pneumoniae isolates in Malaysia

Background

Streptococcus pneumoniae is a major causative agent of severe infections, including sepsis, pneumonia, meningitis, and otitis media, that has since become a major public health concern. In this study, the serotypes distribution of pneumococcal isolates was investigated to predict the efficacy of the 7-valent pneumococcal conjugate vaccine (PCV7) among the Malaysian populations.

Methodology/Principal Findings

A total of 151 clinical isolates were serotyped using multiplex PCR assays. Out of them, there were 21.2% penicillin-resistant, 29.1% penicillin-intermediate, and 49.7% penicillin-susceptible S.pneumoniae strains. Serotypes detected among the Malaysian isolates were 1, 3, 10A, 11A/11D, 12F/12A, 14, 15A, 15B/15C, 16F, 18C/18B/18A/18F, 19A, 19F, 23F, 35B, 35F/47F, 6A/6B, 7C/7B/40, 7F/7A, 9V/9A, and 34. Serotype 19F and 23F were the two most prevalent serotypes detected. Serotypes are highly associated with invasiveness of isolates (p = 0.001) and penicillin susceptibility (p<0.001). Serotype 19F was observed to have increased resistance against penicillin while serotype 19A has high invasive tendency. Age of patients was an important factor underlying the pneumococcal serotypes (p = 0.03) and clinical sites of infections (p<0.001). High prevalence of pneumococcal isolates were detected among children <5 years old at nasopharyngeal sites while elderly adults ≥60 years old were at increased risk for pneumococcal bacteremia.

Conclusion/Significance

Current study revealed that a number of serotypes, especially those associated with high penicillin resistance, have been formulated in the PCV7. Therefore, the protections expected from the routine use of PCV7 would be encouraging for the Malaysian. However, it is not possible to predict serotypes that might become predominant in the future and hence continued surveillance of circulating serotypes will be needed.