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The capacity of Streptococcus pneumoniae to successfully transmit and colonize new human hosts is a critical aspect of pneumococcal population biology and a prerequisite for invasive disease. However, the bacterial mechanisms underlying this process remain largely unknown. To identify bacterial factors required for transmission, we conducted a high-throughput genetic screen with a transposon sequencing (Tn-seq) library of a pneumococcal strain in a ferret transmission model. Key players in both metabolism and transcriptional regulation were identified as required for efficient bacterial transmission. Targeted deletion of the putative C3-degrading protease CppA, iron transporter PiaA, or competence regulatory histidine kinase ComD significantly decreased transmissibility in a mouse model, further validating the screen. Maternal vaccination with recombinant surface-exposed PiaA and CppA alone or in combination blocked transmission in offspring and were more effective than capsule-based vaccines. These data underscore the possibility of targeting pneumococcal transmission as a means of eliminating invasive disease in the population., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Chemical synthesis of conjugate vaccines, consisting of a polysaccharide linked to a protein, can be technically challenging, and in vivo bacterial conjugations (bioconjugations) have emerged as manufacturing alternatives. Bioconjugation relies upon an oligosaccharyltransferase to attach polysaccharides to proteins, but currently employed enzymes are not suitable for the generation of conjugate vaccines when the polysaccharides contain glucose at the reducing end, which is the case for ~75% of Streptococcus pneumoniae capsules. Here, we use an O-linking oligosaccharyltransferase to generate a polyvalent pneumococcal bioconjugate vaccine with polysaccharides containing glucose at their reducing end. In addition, we show that different vaccine carrier proteins can be glycosylated using this system. Pneumococcal bioconjugates are immunogenic, protective and rapidly produced within E. coli using recombinant techniques. These proof-of-principle experiments establish a platform to overcome limitations of other conjugating enzymes enabling the development of bioconjugate vaccines for many important human and animal pathogens.

Streptococcus pneumoniae (the pneumococcus) is a nasopharyngeal commensal and respiratory pathogen. Most isolates express a capsule, the species-wide diversity of which has been immunologically classified into ∼100 serotypes. Capsule polysaccharides have been combined into multivalent vaccines widely used in adults, but the T cell independence of the antibody response means they are not protective in infants. Polysaccharide conjugate vaccines (PCVs) trigger a T cell-dependent response through attaching a carrier protein to capsular polysaccharides. The immune response stimulated by PCVs in infants inhibits carriage of vaccine serotypes (VTs), resulting in population-wide herd immunity. These were replaced in carriage by non-VTs. Nevertheless, PCVs drove reductions in infant pneumococcal disease, due to the lower mean invasiveness of the postvaccination bacterial population; age-varying serotype invasiveness resulted in a smaller reduction in adult disease. Alternative vaccines being tested in trials are designed to provide species-wide protection through stimulating innate and cellular immune responses, alongside antibodies to conserved antigens.

Streptococcus pneumoniae is a major respiratory pathogen that causes millions of deaths worldwide. Although subunit vaccines formulated with the capsular polysaccharides or their protein conjugates are currently-available, low-cost vaccines with wide serotype coverage still remain to be developed, especially for developing countries. Recently, gamma- irradiation has been considered as an effective inactivation method to prepare S. pneumoniae vaccine candidate. In this study, we investigated the immunogenicity and protective immunity of gamma-irradiated S. pneumoniae (r-SP), by comparing with heat-inactivated S. pneumoniae (h-SP) and formalin-inactivated S. pneumoniae (f-SP), both of which were made by traditional inactivation methods. Intranasal immunization of C57BL/6 mice with r-SP in combination with cholera toxin as an adjuvant enhanced S. pneumoniaespecific antibodies on the airway mucosal surface and in sera more potently than that with h-SP or f-SP under the same conditions. In addition, sera from mice immunized with r-SP potently induced opsonophagocytic killing activity more effectively than those of h-SP or f-SP, implying that r-SP could induce protective antibodies. Above all, immunization with r-SP effectively protected mice against S. pneumoniae infection. Collectively, these results suggest that gamma- irradiation is an effective method for the development of a killed whole cell pneumococcal vaccine that elicits robust mucosal and systemic immune responses.

Streptococcus pneumoniae is the leading cause of bacterial pneumonia. Although this is a vaccine preventable disease, S. pneumoniae still causes over 1 million deaths per year, mainly in children under the age of five. The biggest disease burden is in the developing world, which is mainly due to unavailability of vaccines due to their high costs. Protein polysaccharide conjugate vaccines are given routinely in the developed world to children to induce a protective antibody response against S. pneumoniae. One of these vaccines is Prevnar13, which targets 13 of the 95 known capsular types. Current vaccine production requires growth of large amounts of the 13 serotypes, and isolation of the capsular polysaccharide that is then chemically coupled to a protein, such as the diphtheria toxoid CRM 197, in a multistep expensive procedure. In this study, we design, purify and produce novel recombinant pneumococcal protein polysaccharide conjugate vaccines in Escherichia coli, which act as mini factories for the low-cost production of conjugate vaccines. Recombinant vaccine efficacy was tested in a murine model of pneumococcal pneumonia; ability to protect against invasive disease was compared to that of Prevnar13. This study provides the first proof of principle that protein polysaccharide conjugate vaccines produced in E. coli can be used to prevent pneumococcal infection. Vaccines produced in this manner may provide a low-cost alternative to the current vaccine production methodology., (Copyright © 2018. Published by Elsevier Ltd.)

The lack of reliable diagnostic tests for detecting vaccine serotype pneumococcal pneumonia (VTPP) remains a challenging issue in pneumococcal vaccine studies. This study assessed the performances of high-throughput nanofluidic PCR-based pneumococcal serotyping and quantification assay methods using sputum samples (the nanofluidic sputum quantitative PCR [Sp-qPCR] assay) to diagnose 13-valent pneumococcal conjugate VTPP compared with the performance of the serotype-specific urinary antigen detection (UAD) assay using urine samples. Adult pneumonia patients from Japan were enrolled in this study between September 2012 and August 2014. Sputum samples were subjected to the nanofluidic Sp-qPCR assay, quantitatively cultured, and serotyped by the Quellung reaction (SpQt). Urine samples were tested by the UAD method. The diagnostic performances of these tests were assessed using composite reference standards and Bayesian latent class models (BLCMs). Among 244 total patients, 27 (11.1%) tested positive with the UAD assay, while 16 (6.6%) and 34 (13.9%) tested positive with the SpQt and nanofluidic Sp-qPCR assays, respectively, with a cutoff value of ≥10 4 DNA copies/ml, which showed the maximum value of the Youden index. Using BLCMs, the estimated prevalence for VTPP was 12.9%, and the nanofluidic Sp-qPCR assay demonstrated the best performance (sensitivity, 90.2%; specificity, 96.9%), followed by UAD (sensitivity, 75.6%; specificity, 97.9%) and SpQt (sensitivity, 45.8%; specificity, 99.5%). However, when a higher cutoff value of ≥10 7 DNA copies/ml was applied, the performance of UAD became comparable to that of Sp-qPCR. The vaccine serotype-specific pneumococcal DNA load in sputum among UAD-positive patients was 3 logs higher than that among UAD-negative patients ( P = 0.036). The nanofluidic Sp-qPCR assay may be accurate and useful for detecting VTPP among adults., (Copyright © 2018 Kakiuchi et al.)

Purpose of Review: Pneumococcal meningitis is the most frequent form of bacterial meningitis in Europe and the United States. Although early antimicrobial and adjuvant therapy with dexamethasone have helped to improve disease outcome in adults, mortality and morbidity rates remain unsatisfactorily high, emphasizing the need for additional treatment options. Promising targets for adjuvant therapy have been identified recently and will be the focus of this review., Recent Findings: Brain disease in pneumococcal meningitis is caused by direct bacterial toxicity and excessive meningeal inflammation. Accordingly, promising targets for adjuvant therapy comprise limiting the release of toxic bacterial products and suppressing inflammation in a way that maximally protects against tissue injury without hampering pathogen eradication by antibiotics. Among the agents tested so far in experimental models, complement inhibitors, matrix-metalloproteinase inhibitors, and nonbacteriolytic antibiotics or a combination of the above have the potential to more efficiently protect the brain either alone (e.g., in children and outside the high-income settings) or in addition to adjuvant dexamethasone. Additionally, new protein-based pneumococcal vaccines are being developed that promise to improve disease prevention, namely by addressing the increasing problem of serotype replacement seen with pneumococcal conjugate vaccines., Summary: Pneumococcal meningitis remains a life-threatening disease requiring early antibiotic and targeted anti-inflammatory therapy. New adjuvant therapies showed promising results in animal models but need systematic clinical testing.

Molecular properties of bacterial polysaccharides and protein-polysaccharide conjugates play an important role in the efficiency and immunogenicity of the final vaccine product. Size exclusion chromatography (SEC) is commonly used to analyze and characterize biopolymers, including capsular polysaccharides. The objective of this work was to determine the effects of solution ionic strength and pH on the SEC retention of several capsular polysaccharides from S. pneumoniae bacteria in their native and conjugated forms. The retention time of the charged polysaccharides increased with increasing ionic strength and decreasing pH due to compaction of the polysaccharides associated with a reduction in the intramolecular electrostatic interactions. The calculated radius of gyration was in good agreement with model calculations based on the worm-like chain model accounting for the increase in chain stiffness and excluded volume of the charged polysaccharide at low ionic strength. These results provide important insights into the effects of solution ionic strength on physical properties and SEC behavior of capsular polysaccharides and their corresponding conjugates., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The World Health Organization has indicated that we are entering into a post-antibiotic era in which infections that were routinely and successfully treated with antibiotics can now be lethal due to the global dissemination of multidrug resistant strains. Conjugate vaccines are an effective way to create a long-lasting immune response against bacteria. However, these vaccines present many drawbacks such as slow development, high price, and batch-to-batch inconsistencies. Alternate approaches for vaccine development are urgently needed. Here we present a new vaccine consisting of glycoengineered outer membrane vesicles (geOMVs). This platform exploits the fact that the initial steps in the biosynthesis of most bacterial glycans are similar. Therefore, it is possible to easily engineer non-pathogenic Escherichia coli lab strains to produce geOMVs displaying the glycan of the pathogen of interest. In this work we demonstrate the versatility of this platform by showing the efficacy of geOMVs as vaccines against Streptococcus pneumoniae in mice, and against Campylobacter jejuni in chicken. This cost-effective platform could be employed to generate vaccines to prevent infections caused by a wide variety of microbial agents in human and animals.

Vaccines are derived from a variety of sources including tissue extracts, bacterial cells, virus particles, recombinant mammalian, yeast and insect cell produced proteins and nucleic acids. The most common method of vaccine production is based on an initial fermentation process followed by purification. Production of vaccines is a complex process involving many different steps and processes. Selection of the appropriate purification method is critical to achieving desired purity of the final product. Clarification of vaccines is a critical step that strongly impacts product recovery and subsequent downstream purification. There are several technologies that can be applied for vaccine clarification. Selection of a harvesting method and equipment depends on the type of cells, product being harvested, and properties of the process fluids. These techniques include membrane filtration (microfiltration, tangential-flow filtration), centrifugation, and depth filtration (normal flow filtration). Historically vaccine harvest clarification was usually achieved by centrifugation followed by depth filtration. Recently membrane based technologies have gained prominence in vaccine clarification. The increasing use of single-use technologies in upstream processes necessitated a shift in harvest strategies. This review offers a comprehensive view on different membrane based technologies and their application in vaccine clarification, outlines the challenges involved and presents the current state of best practices in the clarification of vaccines., (Copyright © 2015 Elsevier Inc. All rights reserved.)

All currently available Streptococcus pneumoniae (Spn) vaccines have limitations due to their capsular serotype composition. Both the 23-valent Spn polysaccharide vaccine (PPV) and 7, 10, or 13-valent Spn conjugate vaccines (PCV-7, 10, -13) are serotype-based vaccines and therefore they elicit only serotype-specific immunity. Emergence of replacement Spn strains expressing other serotypes has consistently occurred following introduction of capsular serotype based Spn vaccines. Furthermore, capsular polysaccharide vaccines are less effective in protection against non-bacteremic pneumonia and acute otitis media (AOM) than against invasive pneumococcal disease (IPD). These shortcomings of capsular polysaccharide-based Spn vaccines have created high interest in development of non-serotype specific protein-based vaccines that could be effective in preventing both IPD and non-IPD infections. This review discusses the progress to date on development of Spn protein vaccine candidates that are highly conserved by all Spn strains, are highly conserved, exhibit maximal antigenicity and minimal reactogenicity to replace or complement the current capsule-based vaccines. Key to development of a protein based Spn vaccine is an understanding of Spn pathogenesis. Based on pathogenesis, a protein-based Spn vaccine should include one or more ingredients that reduce NP colonization below a pathogenic inoculum. Elimination of all Spn colonization may not be achievable or even advisable. The level of expression of a target protein antigen during pathogenesis is another key to the success of protein based vaccines.. As with virtually all currently licensed vaccines, production of a serum antibody response in response to protein based vaccines is anticipated to provide protection from Spn infections. A significant advantage that protein vaccine formulations can offer over capsule based vaccination is their potential benefits associated with natural priming and boosting to all strains of Spn. One of the most universal and comprehensive approaches of identifying novel vaccine candidates is the investigation of human sera from different disease stages of natural infections. Antigens that are robustly reactive in preliminary human serum screening constitute a pathogen-specific antigenome. This strategy has identified a number of Spn protein vaccine candidates that are moving forward in human clinical trials.

The success of Gavi, the Vaccine Alliance depends on the vaccine markets providing appropriate, affordable vaccines at sufficient and reliable quantities. Gavi's current supplier base for new and underutilized vaccines, such as the human papillomavirus (HPV), rotavirus, and the pneumococcal conjugate vaccine is very small. There is growing concern that following globalization of laws on intellectual property rights (IPRs) through trade agreements, IPRs are impeding new manufacturers from entering the market with competing vaccines. This article examines the extent to which IPRs, specifically patents, can create such obstacles, in particular for developing country vaccine manufacturers (DCVMs). Through building patent landscapes in Brazil, China, and India and interviews with manufacturers and experts in the field, we found intense patenting activity for the HPV and pneumococcal vaccines that could potentially delay the entry of new manufacturers. Increased transparency around patenting of vaccine technologies, stricter patentability criteria suited for local development needs and strengthening of IPRs management capabilities where relevant, may help reduce impediments to market entry for new manufacturers and ensure a competitive supplier base for quality vaccines at sustainably low prices., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Despite the efforts to expand the availability of conjugate vaccines, pneumococcal diseases still pose an enormous burden worldwide. Therefore, several proteins have been investigated as alternative vaccines, alone or in combination with other antigens. With an increasing array of techniques, many of which arose from the publication of the bacterial genome, several proteins have been identified as potential vaccine candidates, and some have even progressed to clinical trials. Also, whole cell vaccines are being studied for the induction of broad ranging protective responses. Here, we briefly summarize the current knowledge on pneumococcal proteins that are being investigated as potential vaccine candidates against pneumococcal infections, and provide an insight on the future generation of protein-based vaccines against Streptococcus pneumoniae.

The efforts were focused on exploring alternative pneumococcal vaccine strategies, aimed at addressing the shortcomings of existing formulations, without compromising efficacy. Our strategy involved the use of the carrier protein, pneumococcal surface protein A (PspA), conjugated with capsular polysaccharides (CPS), to provide effective and non-serotype-dependent protection. In this study, we generated a stable Escherichia coli construct expressing functional PspA from a capsular serotype 6B strain and confirmed it belonging to family 1, which was conjugated with CPS. The distribution of anti-CPS antibody response was almost completely of IgG2a subclass followed by IgG3 and low level of IgG1 subclass, but that of anti-PspA IgG subclass antibodies was almost equal IgG1 and IgG2a subclasses. Though PspA was less conspicuous on the surface of pneumococci than the capsule, the antibodies induced with CPS-rPspA conjugate possessed more accessibility to the surface of Streptococcus pneumoniae serotype 6B and 19F (the same family 1 PspA). By survival experiment, the result suggested that the level of cross-protection after immunized with the conjugate was more measurable within the same family 1. The CPS-rPspA conjugate not only induced CPS-specific protection but also provided PspA specific cross-protection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Streptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis and meningitis. Surface accessible proteins of S. pneumoniae are being explored for the development of a protein-based vaccine in order to overcome the limitations of existing polysaccharide-based pneumococcal vaccines. To identify a potential vaccine candidate, we resolved surface-associated proteins of S. pneumoniae TIGR4 strain using two-dimensional gel electrophoresis followed by immunoblotting with antisera generated against whole heat-killed TIGR4. Ten immunoreactive spots were identified by mass spectrometric analysis that included a putative lipoprotein SP0845. Analysis of the inferred amino acid sequence of sp0845 homologues from 36 pneumococcal strains indicated that SP0845 was highly conserved (>98% identity) and showed less than 11% identity with any human protein. Our bioinformatic and functional analyses demonstrated that SP0845 is the substrate-binding protein of an ATP-binding cassette (ABC) transporter that is involved in nucleoside uptake with cytidine, uridine, guanosine and inosine as the preferred substrates. Deletion of the gene encoding SP0845 renders pneumococci avirulent suggesting that it is essential for virulence. Immunoblot analysis suggested that SP0845 is expressed in in vitro grown pneumococci and during mice infection. Immunofluorescence microscopy and flow cytometry data indicated that SP0845 is surface exposed in encapsulated strains and accessible to antibodies. Subcutaneous immunization with recombinant SP0845 induced high titer antibodies in mice. Hyperimmune sera raised against SP0845 promoted killing of encapsulated pneumococcal strains in a blood bactericidal assay. Immunization with SP0845 protected mice from intraperitoneal challenge with heterologous pneumococcal serotypes. Based on its surface accessibility, role in virulence and ability to elicit protective immunity, we propose that SP0845 may be a potential candidate for a protein-based pneumococcal vaccine.

Pneumococci are capable of vaccine escape by genetic recombination at the targeted capsular locus, significantly reducing long-term vaccine effectiveness. Recently, efforts have been redirected to understanding pneumococcal biology related to potential next-generation vaccine candidates. A variety of serotype-independent protein antigens capable of inducing protective immune responses in tissue culture and animal models of infection have been identified. However, ideal vaccine candidates that are conserved across all genotypes, provide broad population coverage, and induce T-cell dependent immune responses are still under investigation. We examined whether immune responses due to the highly polymorphic CbpA antigen are due to a conserved domain capable of evoking specific immune "memory" across all genotypes of pneumococci. We defined the genotypes in a global dataset of 213 pneumococcal isolates. This isolate collection was genotypically diverse and ideal for establishing the presence of conserved CbpA epitopes as potential protein vaccine candidates. Examination of the CbpA locus sequence was highly polymorphic at both the nucleic acid and amino acid level. Despite this high polymorphism some domains are broadly conserved and consist of different amino acid residues with the same physicochemical properties, and therefore have similar tertiary structures. The two most common domains identified in the CbpA gene are modular teichoic acid phosphorylcholine esterase Pce (2bib:A), and R2 domain (1w9r:A). These conserved domains are immunogenic, therefore capable of inducing long-term host immune responses; moreover they are extracellularly located and thus accessible. We proposed their evaluation as suitable next-generation CbpA-fusion protein vaccine candidates.

Streptococcus pneumoniae is a major cause of morbidity and mortality among children under 5 years of age worldwide. Vaccines have long been used for protection against pneumococcal infections. Capsular polysaccharides of pneumococci are main antigenic components of these vaccines. However, pneumococcal polysaccharide-based vaccines are not able to elicit appropriate immunological responses in young children and cannot induce the immune memory. Thus, pneumococcal conjugate vaccines were developed through chemical coupling of an immunogenic carrier protein to the capsule. The currently available pneumococcal conjugate vaccines elicited protection against the bacterium efficiently. However, these vaccines are expensive to manufacture and have limited serotype coverage. In this mini-review, therefore, we describe approaches attempted by researchers to circumvent the shortcomings of the conjugate vaccines including specifying appropriate cultivation conditions for the production of S. pneumoniae capsular antigens, development of suitable expression systems for the frequently used carrier protein in the conjugate vaccines (cross-reacting material 197), construction of protein-based vaccines, whole-cell vaccines, DNA vaccines, and using antigen delivery vehicles. Future trends in this field are also discussed., (© 2014 S. Karger AG, Basel.)

Pneumococcal conjugate vaccines (PCVs) differ in polysaccharide (PS) dose, carrier protein and conjugation method. PCV development proceeded initially upon principles successfully proven in Haemophilus influenzae type b (Hib) conjugate vaccine development. However, the need to successfully incorporate multiple serotypes while minimizing the total PS dose and total carrier protein load saw some early vaccine candidates fail. Dose-range studies of individual serotypes indicated that much lower PS doses were needed compared with Hib conjugate vaccines, although subsequent studies confirmed that lower Hib PS doses were possible. Furthermore, the immune response to individual serotype doses was carrier protein dependent. A 'one-size fits most' approach has characterized PS dose selection, but peculiarities of individual serotypes are increasingly apparent, raising the question whether re-formulation of PCVs to maximize individual serotype performance is needed.

Streptococcus pneumoniae is a serious Gram-positive pathogen responsible for several life-threatening pneumococcal diseases. Pneumococcal capsular polysaccharide (CPS) is a key virulence determinant of S. pneumoniae and its immunogenicity can be improved by conjugation with a carrier protein. Reductive amination, the most widely used approach for pneumococcal CPS conjugate vaccine (PCV), suffers from low conjugation efficiency and the problem of steric hindrance. Here, copper-catalyzed azide-alkyne cycloaddition was used for development of PCV with long spacer arm (L-PCV). Tetanus toxoid (TT) was used as the carrier protein. The long spacer arm in L-PCV can minimize the problem of steric hindrance between CPS and TT, thereby improving the CPS-specific antibody titers in the mice model. L-PCV can also induce high avidity functional antibody and elicit immunological memory in response to the native CPS., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Streptococcus pneumoniae (the pneumococcus) is the etiologic agent of community-acquired pneumonia and invasive pneumococcal diseases such as septicemia and bacterial meningitis. The increasing antibiotic resistance and the suboptimal efficacy or limited serotype coverage of currently available vaccines urgently requires novel approaches in exploring new antimicrobials, therapeutic intervention strategies and vaccines. The current vaccine development strategies rely on the hypothesis that surface-exposed proteins, which are essential for pneumococcal virulence, are the most suitable candidates for future protein-based vaccines. Since virulence is closely linked with bacterial fitness, the potential of a pathogen to colonize and infect the host depends further on its physiology. This review summarizes the application of genome-wide techniques and their exploitation to decipher fundamental insights into bacterial factors associated with fitness, metabolism and virulence, leading to the discovery of vaccine candidates or antimicrobials.

Aim: Study protective activity of S. pneumoniae protein-containing antigen complex obtained from T3No.3 strain against infection by homologous pneumococcus strain., Materials and Methods: S. pneumoniae T3No.3 (serotype 3) strain obtained from collection of pneumococcus strains of Mechnikov Research Institute of Vaccines and Sera was used in the study. S. pneumoniae protein-containing antigen complex was isolated by precipitation by 2 volumes of acetone of supernatant fraction of cultural medium used for pneumococcus cultivation. Molecular mass of proteins contained in S. pneumoniae antigen complex was determined by SDS electrophoresis in polyacrylamide gel. Protective activity of S. pneumoniae protein-containing antigen complex was studied in BALB/c line mice active protection experiments. Activity of mice immune sera obtained against whole-cell pneumococcus culture (T3No.3 strain) was determined in vitro by solid phase indirect EIA., Results: The data obtained give evidence that the isolated protein-containing antigen complex from S. pneumoniae T3No.3 strain effectively protects mice from consequent infection by a homologous S. pneumoniae strain. S. pneumoniae protein-containing antigen complex sorbed on solid phase at 5 microg dose was established by using EIA to interact with homologous mice immune sera., Conclusion: The results of the carried out studies allow to move to studies of cross-activity of S. pneumoniae protein-containing antigen complex isolated from T3No.3 strain.

Knowledge of co-colonization with multiple pneumococcal serotypes is becoming very important in the light of both serotype replacement and switching as a result of vaccination. Co-colonization has been reported to occur in up to 30% of carriers, especially in populations with high Streptococcus pneumoniae carriage rates. For the determination of co-colonization, single colonies of nasopharyngeal specimens are serotyped with the Quellung method, a costly method with a low sensitivity. Here we explore the use of a multiplex PCR to identify simultaneous carriage of the capsular serotypes targeted by the 7-valent conjugate vaccine. We applied this multiplex PCR to 50 primary cultures from the nasopharyngeal swabs of healthy Warao Amerindian children, a population with a high pneumococcal carriage rate, most of them with vaccine serotypes, and we identified a second serotype in 20% (n=10) of the pneumococci carriers. These results were confirmed by detailed serotyping of multiple colonies isolated from the primary culture with the Quellung method. We conclude that the multiplex PCR is a sensitive, simple and cost-effective method for detecting multiple serotypes in nasopharyngeal cultures, and thus might be useful for the monitoring of pneumococcal colonization over time, especially in the surveillance of nasopharyngeal colonization after conjugate vaccination.

Here we describe a novel method to conjugate pneumococcal polysaccharides (PnPS) to Luminex microspheres for use in serological assays. 4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methyl-morpholinium (DMTMM) modification of PnPS and conjugation to carboxyl functional groups on Luminex microspheres (COOH-DMTMM method) was shown to be a reproducible chemistry that efficiently conjugated PnPS to Luminex microspheres without affecting the antigenicity of a broad set of PnPS. The COOH-DMTMM method was compared to three other methods for robustness, reproducibility and effect on PnPS antigenicity in a multiplexed assay format. The other methods examined included adsorption of the unmodified PnPS to Luminex microspheres, oxidation of the PnPS to conjugate them to amino-modified microspheres using carbodiimide chemistry and poly-l-lysine modification of the PnPS before conjugating to carboxy Luminex microspheres using carbodiimide chemistry. Of the four methods, the COOH-DMTMM chemistry was shown to be a robust methodology, producing stable PnPS coupled microspheres with a 4-log dynamic range and low cross-reactivity when used in a PnPS-specific IgG serology assay. This novel chemistry should be useful for developing serological assays to measure antibodies to polysaccharides for use in vaccine and epidemiology studies.

Streptococcus pneumoniae is a leading cause of bacterial pneumonia, meningitis, and acute otitis media in children and adults worldwide. According to World Health Organization estimates, at least 1 million children under 5 years of age die each year from pneumococcal pneumonia. The emergence of resistant strains necessitates the development of an effective vaccine with a large serotype coverage. The 11 most common serotypes cause 72-83% of all serious pneumococcal diseases worldwide. Currently marketed 23-valent pneumococcal polysaccharide vaccine provides large serotype coverage and offers a less expensive option. However, it is efficacious only in adults but not in infants. Conjugate vaccines offer a solution by generating immunological memory already at early age. A recently licensed 7-valent conjugate vaccine is immunogenic and efficacious in infants. Its serotype coverage might be sufficient in Europe and North America, but not in Africa, Asia and Oceania. A need exists to develop pneumococcal vaccines with lower cost and larger serotype coverage. Several 11-valent pneumococcal conjugate vaccines are being evaluated in phase I-III trials. This study reviews the current state of pneumococcal problem and pneumococcal vaccines in clinical use.