Multivalent Pneumococcal Conjugate Vaccines Research Articles

Streptococcus pneumoniae serotypes 8 and 22F causing invasive pneumococcal disease in the Czech Republic in 2014-2020: whole genome sequencing (WGS) analysis.

An analysis is presented of whole genome data of Streptococcus pneumoniae serotypes 8 and 22F isolated in the Czech Republic from invasive pneumococcal disease (IPD) in 2014-2020. New multivalent pneumococcal conjugate vaccines (PCVs) are effective against these serotypes. Recently, serotypes 8 and 22F have been among the leading causes of IPD in the Czech Republic.S. pneumoniae isolates from the Czech Republic were compared with those of the same serotypes recovered in other countries in the same period and available in the international database PubMLST. Isolates from IPD of serotypes 8 (22 isolates) and 22F (21 isolates) recovered in the Czech Republic in 2014-2020 were subjected to whole genome sequencing (WGS). The genomes were analysed and compared using the international database PubMLST. Most of the studied Czech serotype 8 isolates belong to two main subpopulations. The first subpopulation, dominated by ST-53 isolates, is part of a highly abundant group of genetically close European and non-European isolates that are clearly separated on the phylogenetic network. The second subpopulation of Czech serotype 8 isolates (dominated by ST-404) is more genetically variable and forms a separate lineage on the global phylogenetic network, with no other European isolates. Czech isolates of serotype 22F are a homogeneous population with a clear predominance of ST-433, which belongs to a genetically close European population. The analysis of WGS data of IPD isolates of serotypes 8 and 22F provided a detailed insight into the genetic relationships between the Czech populations of these serotypes. It also allowed comparison of the Czech populations with the matched populations from other European and non-European countries. The obtained results add to the body of knowledge about the spread of genetic lineages causing IPD in the Czech Republic in the post-vaccination period and provide a basis for considering whether the use of the new multivalent PCVs in the Czech Republic would be beneficial.

Individual polysaccharide quantification in polyvalent pneumococcal conjugate vaccine using rate nephelometry

The multivalent pneumococcal conjugate vaccine (PCV) contains purified polysaccharides of different serotypes conjugated to a carrier protein. Testing the final formulated product for individual serotype polysaccharide content is critical in vaccine quality control which requires an assay specific to each serotype polysaccharide present in the formulated product. Antibodies specific to the serotypes specific polysaccharide were used in rate nephelometry assay for quantifying individual serotype polysaccharides in the formulated vaccine. Generally, native polysaccharide (NP) have been used as reference standard. However, the polysaccharide antigen in the vaccine product is in the conjugate form (CRM197 linked) and hence using NP as a reference standard may not be suitable. Activated quenched polysaccharide (AQP) as a reference standard in rate nephelometry would be more appropriate. The epitope structure of AQP closely represents the polysaccharide-protein conjugate drug product (DP) after trypsin digestion. Hence, AQP was evaluated as a novel reference standard for the accurate and precise determination of individual polysaccharides in the multivalent DP. Rate nephelometry assay using AQP could be used for DP release and stability for monitoring time-dependent changes in the product and establishing the shelf life. A similar strategy could be applied to test and release monovalent or multivalent polysaccharide-protein conjugate vaccines (Meningococcal, Haemophilus influenza Type B, Typhoidal, and non-typhoidal salmonella).

Safety and immunogenicity of a multivalent pneumococcal conjugate vaccine given with 13-valent pneumococcal conjugate vaccine in healthy infants: A phase 2 randomized trial

ABSTRACT Use of pneumococcal conjugate vaccines (PCVs) has led to substantial reductions in the global burden of pediatric pneumococcal disease. Expansion of serotype coverage has been achieved by increasing PCV valency, but this may carry the potential risk of antibody interference. A complementary 7-valent PCV (cPCV7) including polysaccharide conjugates from 7 non–13-valent (PCV13) serotypes was developed to potentially complement PCV13-mediated protection and expand serotype coverage. This study evaluated cPCV7 and PCV13 coadministered in separate limbs or separated in time in infants. This phase 2, multicenter, open-label study included 512 infants randomized 1:1:1 to receive cPCV7 coadministered with PCV13 at ages 2, 4, 6, and 12 months (cPCV7 Coadministered); cPCV7 given at ages 3, 5, 7, and 13 months, 3‒5 weeks after PCV13 (cPCV7 Separated); or PCV13 at ages 2, 4, 6, and 12 months followed by a single supplemental dose of cPCV7 at 13 months (PCV13 Control). Safety evaluations included local reactions, systemic events, and adverse events. Serotype-specific immunoglobulin G concentrations and opsonophagocytic activity titers were assessed. The safety profile of cPCV7 was similar to that of PCV13. cPCV7 was well-tolerated in infants when coadministered with or given separately from PCV13. Robust and functional immune responses for all cPCV7 serotypes were observed in both cPCV7 groups. No immunologic interference was observed for either the cPCV7 or PCV13 serotypes with coadministration. A single cPCV7 dose induced immune responses in toddlers. These findings support potential coadministration of a complementary PCV to supplement protection provided by existing PCVs. Trial registration: ClinicalTrials.gov, NCT03550313.

Antibody enhanced HPLC for serotype-specific quantitation of polysaccharides in pneumococcal conjugate vaccine

Bacterial infection remains as one of the major healthcare issues, despite significant scientific and medical progress in this field. Infection by Streptococcus Pneumoniae (S. Pneumoniae) can cause pneumonia and other serious infectious diseases, such as bacteremia, sinusitis and meningitis. The pneumococcal capsular polysaccharides (CPS) that constitute the outermost layer of the bacterial cell are the main immunogens and protect the pathogen from host defense mechanisms. Over 90 pneumococcal CPS serotypes have been identified, among which more than 30 can cause invasive pneumococcal diseases that could lead to morbidity and mortality. Multivalent pneumococcal vaccines have been developed to prevent diseases caused by S. Pneumoniae. These vaccines employ either purified pneumococcal CPSs or protein conjugates of these CPSs to generate antigen-specific immune responses for patient protection. Serotype-specific quantitation of these polysaccharides (Ps) antigen species are required for vaccine clinical dosage, product release and quality control. Herein, we have developed an antibody-enhanced high-performance liquid chromatography (HPLC) assay for serotype-specific quantitation of the polysaccharide contents in multivalent pneumococcal conjugate vaccines (PCVs). A fluorescence-labeled multiplex assay format has also been developed. This work laid the foundation for a serotype-specific antigen assay format that could play an important role for future vaccine research and development.

Characterization of High Molecular Weight Pneumococcal Conjugate by SEC-MALS and AF4-MALS.

Infections by Streptococcus pneumoniae can cause serious pneumococcal diseases and other medical complications among patients. Polysaccharide-based vaccines have been successfully developed as prophylactic agents against such deadly bacterial infections. In the 1980s, PNEUMOVAX® 23 were introduced as the first pneumococcal polysaccharide vaccines (PPSV). Later, pneumococcal polysaccharides were conjugated to a carrier protein to improve immune responses. Pneumococcal conjugate vaccines (PCV) such as PREVNAR® and VAXNEUVANCE™ have been developed. Of the more than 90 pneumococcal bacteria serotypes, serotype 1 (ST-1) and serotype 4 (ST-4) are the two main types that cause invasive pneumococcal diseases (IPD) that could lead to morbidity and mortality. Development of a novel multi-valent PCV against these serotypes requires extensive biophysical and biochemical characterizations of each monovalent conjugate (MVC) in the vaccine. To understand and characterize these high molecular weight (Mw) polysaccharide protein conjugates, we employed the multi-angle light scattering (MALS) technique coupled with size-exclusion chromatography (SEC) separation and asymmetrical flow field flow fractionation (AF4). MALS analysis of MVCs from the two orthogonal separation mechanisms helps shed light on the heterogeneity in conformation and aggregation states of each conjugate.

Pneumococcal Surface Proteins as Virulence Factors, Immunogens, and Conserved Vaccine Targets.

Streptococcus pneumoniae is an opportunistic pathogen that causes over 1 million deaths annually despite the availability of several multivalent pneumococcal conjugate vaccines (PCVs). Due to the limitations surrounding PCVs along with an evolutionary rise in antibiotic-resistant and unencapsulated strains, conserved immunogenic proteins as vaccine targets continue to be an important field of study for pneumococcal disease prevention. In this review, we provide an overview of multiple classes of conserved surface proteins that have been studied for their contribution to pneumococcal virulence. Furthermore, we discuss the immune responses observed in response to these proteins and their promise as vaccine targets.

A randomized phase 1/2 study of the safety and immunogenicity of a multivalent pneumococcal conjugate vaccine in healthy adults 50 through 85 years of age

ABSTRACT Pneumococcal disease can be serious and debilitating in older adults. Pneumococcal conjugate vaccines (PCVs), such as the 13-valent PCV (PCV13), reduce pneumococcal disease rates caused by vaccine serotypes. Development of PCVs offering additional coverage against serotypes not contained in PCV13 can reduce disease burden further. The complementary 7-valent PCV (cPCV7) contains seven non-PCV13 serotypes (8, 10A, 11A, 12F, 15B, 22F, 33F) and can expand coverage by supplementing direct or indirect protection from existing PCVs. This phase 1/2, randomized, active-controlled, observer-blinded study evaluated cPCV7 safety and immunogenicity in healthy adults 50–85 years of age. Stage 1 randomized 66 healthy adults (50–64 years) naive to pneumococcal vaccines to receive cPCV7 or licensed tetanus, diphtheria, and acellular pertussis vaccine; Stage 2 randomized 445 healthy adults (65–85 years) previously vaccinated with PCV13 to receive cPCV7 or 23-valent polysaccharide vaccine. Local reactions and systemic events up to 14 days and adverse events (AEs) through 1 month after vaccination were assessed. Immunogenicity was evaluated by serotype-specific opsonophagocytic activity (OPA) assays before and 1 month after vaccination (and after 12 months in Stage 2). Rates of local reactions, systemic events, and AEs were generally similar after receipt of cPCV7 or control. Robust OPA responses were observed for all seven serotypes 1 month after cPCV7; titers declined yet remained above baseline 12 months after vaccination. Overall, this study found that in adults ≥50 years of age, cPCV7 was safe, well tolerated, and elicited functional immune responses to vaccine serotypes. ClinicalTrials.gov: NCT03313050

Pneumococcal conjugate vaccines for preventing otitis media

Acute otitis media (AOM) is a very common respiratory infection in early infancy and childhood. The marginal benefits of antibiotics for AOM in low-risk populations in general, the increasing problem of bacterial resistance to antibiotics and the huge estimated direct and indirect annual costs associated with otitis media (OM) have prompted a search for effective vaccines to prevent AOM. To assess the effect of pneumococcal conjugate vaccines (PCVs) in preventing AOM in children up to 12 years of age. We searched CENTRAL (2013, Issue 11), MEDLINE (1995 to November week 3, 2013), EMBASE (1995 to December 2013), CINAHL (2007 to December 2013), LILACS (2007 to December 2013) and Web of Science (2007 to December 2013). Randomised controlled trials (RCTs) of PCVs to prevent AOM in children aged 12 years or younger, with a follow-up of at least six months after vaccination. Two review authors independently assessed trial quality and extracted data. We included 11 publications of nine RCTs (n = 48,426 children, range 74 to 37,868 per study) of 7- to 11-valent PCV (with different carrier proteins). Five trials (n = 47,108) included infants, while four trials (n = 1318) included children aged one to seven years that were either healthy (one study, n = 264) or had a previous history of upper respiratory tract infection (URTI), including AOM. We judged the methodological quality of the included studies to be moderate to high. There was considerable clinical diversity between studies in terms of study population, type of conjugate vaccine and outcome measures. We therefore refrained from pooling the results.In three studies, the 7-valent PCV with CRM197 as carrier protein (CRM197-PCV7) administered during early infancy was associated with a relative risk reduction (RRR) of all-cause AOM ranging from -5% in high-risk children (95% confidence interval (CI) -25% to 12%) to 7% in low-risk children (95% CI 4% to 9%). Another 7-valent PCV with the outer membrane protein complex of Neisseria meningitidis (N. meningitidis) serogroup B as carrier protein, administered in infancy, did not reduce overall AOM episodes, while a precursor 11-valent PCV with Haemophilus influenzae (H. influenzae) protein D as carrier protein was associated with a RRR of all-cause AOM episodes of 34% (95% CI 21% to 44%).A 9-valent PCV (with CRM197 carrier protein) administered in healthy toddlers was associated with a RRR of (parent-reported) OM episodes of 17% (95% CI -2% to 33%). CRM197-PCV7 followed by 23-valent pneumococcal polysaccharide vaccination administered after infancy in older children with a history of AOM showed no beneficial effect on first occurrence and later AOM episodes. In a study in older children with a previously diagnosed respiratory tract infection, performed during the influenza season, a trivalent influenza vaccine combined with placebo (TIV/placebo) led to fewer all-cause AOM episodes than vaccination with TIV and PCV7 (TIV/PCV7) when compared to hepatitis B vaccination and placebo (HBV/placebo) (RRR 71%, 95% CI 30% to 88% versus RRR 57%, 95% CI 6% to 80%, respectively) indicating that CRM197-PCV7 after infancy may even have negative effects on AOM. Based on current evidence of the effects of PCVs for preventing AOM, the licensed 7-valent CRM197-PCV7 has modest beneficial effects in healthy infants with a low baseline risk of AOM. Administering PCV7 in high-risk infants, after early infancy and in older children with a history of AOM, appears to have no benefit in preventing further episodes. Currently, several RCTs with different (newly licensed, multivalent) PCVs administered during early infancy are ongoing to establish their effects on AOM. Results of these studies may provide a better understanding of the role of the newly licensed, multivalent PCVs in preventing AOM. Also the impact on AOM of the carrier protein D, as used in certain pneumococcal vaccines, needs to be further established.

An evaluation of emerging vaccines for childhood pneumococcal pneumonia

BackgroundPneumonia is the leading cause of child mortality worldwide. Streptococcus pneumoniae (SP) or pneumococcus is estimated to cause 821,000 child deaths each year. It has over 90 serotypes, of which 7 to 13 serotypes are included in current formulations of pneumococcal conjugate vaccines that are efficacious in young children. To further reduce the burden from SP pneumonia, a vaccine is required that could protect children from a greater diversity of serotypes. Two different types of vaccines against pneumococcal pneumonia are currently at varying stages of development: a multivalent pneumococcal conjugate vaccine covering additional SP serotypes; and a conserved common pneumococcal protein antigen (PPA) vaccine offering protection for all serotypes.MethodsWe used a modified CHNRI methodology for setting priorities in health research investments. This was done in two stages. In Stage I, we systematically reviewed the literature related to emerging SP vaccines relevant to several criteria of interest: answerability; efficacy and effectiveness; cost of development, production and implementation; deliverability, affordability and sustainability; maximum potential for disease burden reduction; acceptability to the end users and health workers; and effect on equity. In Stage II, we conducted an expert opinion exercise by inviting 20 experts (leading basic scientists, international public health researchers, international policy makers and representatives of pharmaceutical companies). The policy makers and industry representatives accepted our invitation on the condition of anonymity, due to sensitive nature of their involvement in such exercises. They answered questions from CHNRI framework and their “collective optimism” towards each criterion was documented on a scale from 0 to 100%.ResultsThe experts expressed very high level of optimism (over 80%) that low-cost polysaccharide conjugate SP vaccines would satisfy each of the 9 relevant CHNRI criteria. The median potential effectiveness of conjugate SP vaccines in reduction of overall childhood pneumonia mortality was predicted to be about 25% (interquartile range 20-38%, min. 15%, max 45%). For low cost, cross-protective common protein vaccines for SP the experts expressed concerns over answerability (72%) and the level of development costs (50%), while the scores for all other criteria were over 80%. The median potential effectiveness of common protein vaccines in reduction of overall childhood pneumonia mortality was predicted to be about 30% (interquartile range 26-40%, min. 20%, max 45%).ConclusionsImproved SP vaccines are a very promising investment that could substantially contribute to reduction of child mortality world-wide.

Pneumococcal serotype 3 otitis media, limited effect of polysaccharide conjugate immunisation and strain characteristics

Background In contrast to the other vaccine serotypes, no protection could be demonstrated in the POET study against serotype 3 acute otitis media (AOM) following primary and booster vaccination with a multi-valent pneumococcal conjugate vaccine. Methods AOM efficacy and immunogenicity data were reviewed. Pheno- and genotypic characteristics of different serotype 3 strains including efficacy study AOM isolates were evaluated. Results Evaluation of vaccine efficacy before and after booster vaccination indicated that lack of efficacy against serotype 3 pneumococci might have been due to declined protection following the booster dose. However, although atypical immunogenicity was observed for serotype 3 in the second year of life, the capacity to respond to serotype 3 plain polysaccharide was not impaired. All but one of the serotype 3 strains examined had abundant polysaccharide capsules. Comparison of serotype 3 capsular polysaccharide biosynthesis gene sequences found no relevant differences between any of the serotype 3 strains, but mRNA transcript levels were lower for the less densely encapsulated strain. Conclusion Lack of clinical efficacy against serotype 3 AOM following pneumococcal conjugate vaccination may be due to an impaired induction of immune memory. A possible alternative explanation may lie with the atypically abundant expression of capsular polysaccharide which could make serotype 3 strains less susceptible to anti-polysaccharide antibody defence mechanisms in the middle ear. The occurrence of acapsular forms during biofilm growth may also play a role. Clinical impact against otitis media, of vaccines containing pneumococcal serotype 3 components, remains unclear until further investigations have demonstrated the value.

Control of pneumococcal disease in the United Kingdom – the start of a new era

In 2000, a multi-valent pneumococcal conjugate vaccine, known as Prevnar, was licensed for use in infants and young children in the USA. The subsequent introduction of the vaccine into the childhood immunization schedule in that country led to a significant decrease in pneumococcal disease. The vaccine is effective against invasive and non-invasive pneumococcal infection, can be used in young children as well as adults and, like all conjugate vaccines, provides long-lasting immunity. Moreover, it reduces the incidence of antibiotic resistance because a number of resistant serotypes are targeted by the vaccine. Prevnar, also known as Prevenar, has since been licensed in numerous countries, including the UK. On 8 February 2006, the Departments of Health in England, Scotland and Wales announced the inclusion of Prevenar in the childhood immunization schedule. This announcement has important implications for pneumococcal infection, disease surveillance and immunization policy in the UK.

Rôle de la vaccination sur les infections invasives à pneumocoque

Streptococcus pneumoniae is a principal cause of serious invasive infections of childhood, like meningitis, septicemia, bacteremia, or bacteraemic pneumonia. Antibodies to the capsular polysaccharide of S. pneumoniae facilitate clearance of pneumococcal organism through phagocytosis. Pneumococcal polysaccharide vaccines, which have been available for decades, unfortunately have limited or little to no efficacy in children younger than two years of age, who are at highest risk. The immunogenicity of bacterial polysaccharides can be significantly improved through conjugation. Covalent link of the polysaccharide to a T-cell dependent immunogen, a protein, has been shown to make the polysaccharide immunogenic in very young infants. Conjugation technology has been used to develop a multivalent pneumococcal conjugate vaccine, including seven serotypes (4, 6B, 9V, 14, 18C, 19F and 23F) selected for their pre-eminent contribution to invasive diseases (IPD). Immunization with this conjugated pneumococcal vaccine confers to infants and young children protection that is long lasting against invasive pneumococcal disease.The clinical development program established that the protective efficacy was 97% against invasive pneumococcal disease caused by vaccine serotypes and was maintained for at least 3.5 years (Northern California Kaiser Permanente study, NCKP). It has been possible to evaluate the large-scale impact of this pneumococcal vaccine since April 2000 when it was introduced in the USA. In children aged less than five years, data show a dramatic fall in invasive disease due to vaccine serotypes within the NCKP system: a decrease over 95% by 2003. Data from the Active Bacterial Core Surveillance (ABCs) Network of the U.S. Centers for Disease Control and Prevention for 1998 to 2001 reveals a reduction of vaccine serotype IPD of 77.7% ( P <0.001) in children less than two years of age during the 18 months following the introduction of Prevenar ®. In both surveillance settings, the data also indicate that, the increase, if any, in invasive disease caused by non-vaccine serotypes is of limited magnitude. Vaccination reduces the nasopharyngeal carriage of vaccine serotypes and decreases the likelihood of transmission of these S. pneumoniae strains from vaccinated children to unvaccinated neighbors, thereby lowering the rates of IPD in the non-vaccinated community (herd protection). Consequently NCKP and ABC s data indicate an overall decrease in IPD among older children and adults (anyone greater than five years of age). In conclusion, the protective efficacy of Prevenar ®, the individual direct protection as well as the indirect herd protection, is well established. Those findings offer promising perspectives for the prevention of pneumococcal infections in France.

PppA, a Surface-Exposed Protein ofStreptococcus pneumoniae, Elicits Cross-Reactive Antibodies That Reduce Colonization in a Murine Intranasal Immunization and Challenge Model

The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the present conjugate vaccine may still cause pneumococcal disease. To address these serotypes, and the remaining otitis media due to Streptococcus pneumoniae, efforts have been devoted to identifying protective protein antigens. Immunity to conserved surface proteins important for adhesion, nutrient acquisition, or other functions could result in a reduction of colonization and a lower disease potential. We have been searching for conserved surface-exposed proteins from S. pneumoniae that may be involved in pathogenesis to test as vaccine candidates. Here, an approximately 20-kDa protein that has significant homology to a nonheme iron-containing ferritin protein from Listeria innocua and other bactoferritins was identified as pneumococcal protective protein A (PppA). We expressed and purified recombinant PppA (rPppA) and evaluated its potential as a vaccine candidate. The antibodies elicited by purified rPppA were cross-reactive with PppA from multiple strains of S. pneumoniae and were directed against surface-exposed epitopes. Intranasal immunization of BALB/c mice with PppA protein and either a synthetic monophosphoryl lipid A analog, RC529AF, or a cholera toxin mutant, CT-E29H, used as an adjuvant reduced nasopharyngeal colonization in mice following intranasal challenge with a heterologous pneumococcal strain. PppA-specific systemic and local immunoglobulin G (IgG) and IgA antibody responses were induced. The antisera reacted with whole cells of a heterologous S. pneumoniae type 3 strain. These observations indicate that PppA may be a promising candidate for inclusion in a vaccine against pneumococcal otitis media.

Pneumonia in children in the developing world: New challenges, new solutions

Pneumonia has been a leading cause of death in both developed and developing countries as long as health indicators have been available. Yet the issues of concern for this syndrome are far from static. Improvements in access to health services have lowered infant mortality rates, benefiting children around the world and lowering the fraction of child deaths caused by pneumonia. However, progress has been interrupted repeatedly by the emergence of new pathogens. Landmark randomized controlled trials have now demonstrated the effectiveness of Haemophilus influenzae type b (Hib) and multivalent pneumococcal conjugate vaccines against childhood pneumonia, as well as meningitis and bacteremic disease. Momentum has gathered to tackle long-standing economic obstacles to expand access to new vaccines and programs for the poorest countries of the world. A pressing challenge for the control of pneumonia in developing countries is to identify better metrics for pneumonia. Surveillance tools are needed that will bridge studies of interventions, establish preventable disease burden, and serve as indicators for monitoring new programs.

Development and Evaluation of Pneumococcal Conjugate Vaccines: Clinical Trials and Control Tests

Streptococcus pneumoniae is a major cause of pneumonia, meningitis, and otitis media and is responsible for disease in young children, the elderly, and immunocompromised individuals. Emerging high-level resistance to penicillin, multiple antibiotics, and tolerance to vancomycin emphasizes the importance of preventing pneumococcal infection by alternative methods such as immunization. The development of pneumococcal conjugate vaccines using the same carrier proteins as those used in Hemophilus influenzae type b vaccines has enhanced the immune response in infants and children compared with polysaccharide vaccines and has significantly improved the ability to prevent pneumococcal disease in this population worldwide. Here we review the clinical trials of multivalent pneumococcal conjugate vaccines under evaluation, identify potential carrier proteins considered for development of future pneumococcal conjugate vaccines, discuss issues regarding licensure of new candidate vaccines from a clinical trial and quality control perspective, and alternative vaccine strategies for the prevention of pneumococcal disease.

Recombinant PhpA protein, a unique histidine motif-containing protein from Streptococcus pneumoniae, protects mice against intranasal pneumococcal challenge.

The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the current conjugate vaccine may still cause pneumococcal disease. To address these serotypes and the remaining otitis media due to Streptococcus pneumoniae, we have been evaluating antigenically conserved proteins from S. pneumoniae as vaccine candidates. A previous report identified a 20-kDa protein with putative human complement C3-proteolytic activity. By utilizing the publicly released pneumococcal genomic sequences, we found the gene encoding the 20-kDa protein to be part of a putative open reading frame of approximately 2,400 bp. We recombinantly expressed a 79-kDa fragment (rPhpA-79) that contains a repeated HxxHxH motif and evaluated it for vaccine potential. The antibodies elicited by the purified rPhpA-79 protein were cross-reactive to proteins from multiple strains of S. pneumoniae and were against surface-exposed epitopes. Immunization with rPhpA-79 protein adjuvanted with monophosphoryl lipid A (for subcutaneous immunization) or a mutant cholera toxin, CT-E29H (for intranasal immunization), protected CBA/N mice against death and bacteremia, as well as reduced nasopharyngeal colonization, following intranasal challenge with a heterologous pneumococcal strain. In contrast, immunization with the 20-kDa portion of the PhpA protein did not protect mice. These results suggest that rPhpA-79 is a potential candidate for use as a vaccine against pneumococcal systemic disease and otitis media.

Serotype of Streptococcus pneumoniae capsular polysaccharide can modify the Th1/Th2 cytokine profile and IgG subclass response to pneumococal-CRM 197 conjugate vaccines in a murine model

The cellular and antibody responses to type 14 and type 19F Streptococcus pneumoniae capsular polysaccharides (PS) conjugated to CRM 197 were investigated in a mouse model developed for pre-clinical evaluation and quality control of pneumococcal conjugate vaccines. Total IgG antibody and IgG subclasses against PS and the carrier protein for both conjugates were measured in addition to the T cell proliferation and cytokine profiles induced by these conjugates. While unconjugated PS 14 and 19F were at best only weakly immunogenic, both types of conjugate induced strong primary and secondary IgG responses to PS. The responses induced by the two conjugates to the carrier protein were very different; a high level of anti-CRM 197 IgG was induced only by the PS19F conjugate whereas a very weak response was induced by the PS14 conjugate. Interestingly, the IgG subclass distribution was different for the two conjugates; for PS19F conjugate, the IgG response was almost completely of IgG1 subclass with low levels of IgG3 and IgG2a while the response to PS14 conjugate was mainly of the IgG1 and IgG2a subclasses with a low level of IgG3. The anti-CRM 197 IgG subclass distribution was identical with that to the corresponding conjugated PS. Both types of conjugate induced strong T cell proliferation to recall antigens but induced different patterns of cytokine response in immune spleen cells which were indicative of a Th0 response or a mixture of Th1 and Th2 responses with a bias towards Th2 response in PS19F-CRM 197 immunised mice. In conclusion, PS14- and PS19F-CRM 197 conjugates induced different IgG subclass patterns as a result of inducing different patterns of cytokine response to the carrier protein. This indicates that the serotype of PS can modify the Th1/Th2 response to the carrier protein, which has a direct effect and can predict the IgG subclass of the PS response. Finally, we conclude that this model appears suitable for studying the immunogenicity and immune interaction of different components of multivalent pneumococcal conjugate vaccines and may be applicable to their pre-clinical evaluation and quality control.

Effect of individual conjugate dose on immunogenicity of type 6B pneumococcal polysaccharide– N. meningitidis outer membrane protein complex conjugate vaccines in infant rhesus monkeys

A pneumococcal conjugate vaccine (PCV) has been developed consisting of capsular polysaccharide (Ps) coupled to the outer membrane protein complex of Neiserria meningitidis serogroup B. Experiments were conducted in infant rhesus monkeys to assess the potential to administer multiple Pn types in a single vaccine. A single type conjugate, 6B, was dosed from 0.025 to 25 μg Ps. Peak anti-6B Ps Ab titers were seen at lower doses of 0.025 and 0.25 μg Ps, while reduced titers of anti-6B Ps Ab were observed at the highest doses of conjugate administered, 2.5 and 25 μg Ps. By mixing free Ps, carrier, or another monovalent PCV with this 6B PCV, it was determined that reduced anti-6B Ps titers at high PCV doses were associated only with the quantity of type-specific Ps in the conjugate. Thus, increasing the amount of carrier protein or adding an additional monovalent conjugate did not significantly affect the response to type 6B Ps. These results suggest that, given an appropriately determined dose per individual pneumococcal Ps type, a multivalent PCV that includes many different types should have satisfactory clinical immunogenicity.

Immunogenicity of pneumococcal conjugate vaccines.

Prevention of pneumococcal infections is a public health priority because of the high impact of the disease and because of the increasing problems due to antimicrobial resistance. Traditional vaccines, consisting of purified capsular polysaccharides (PSs) of Streptococcus pneumoniae, are not immunogenic in young children. In addition they confer only limited protection in patients with immunodeficiencies and hematologic malignancies. Immunogenicity of the PS vaccine has been enhanced by coupling pneumococcal PSs to proteins to produce a conjugate vaccine. Conjugate molecules are designed to possess T cell dependent properties, such as immunogenicity in early infancy, stimulation of high levels of IgG isotype antibodies and enhanced immunologic memory responses. In the clinical studies multivalent pneumococcal conjugate vaccines have been shown to induce an IgG-dominating serum antibody response against common pneumococcal serotypes causing infections in children. A booster dose later in life creates a robust and rapid antibody response, indicating the existence of immunologic memory in primed children. Antibodies induced by conjugate vaccines are functionally active, as demonstrated by their high avidity and opsonophagocytic activity.

CpG oligodeoxynucleotides act as adjuvants for pneumococcal polysaccharide-protein conjugate vaccines and enhance antipolysaccharide immunoglobulin G2a (IgG2a) and IgG3 antibodies.

Pneumococcal polysaccharide-protein conjugate vaccines elicit antipolysaccharide antibodies, but multiple doses are required to achieve protective antibody levels in children. In addition, the immunogenicity of experimental multivalent pneumococcal conjugate vaccines varies with different polysaccharide serotypes. One strategy to improve these vaccines is to incorporate an adjuvant to enhance their immunogenicity. Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are adjuvants that promote T-cell and T-dependent antibody responses to protein antigens, but it has been unclear whether CpG ODN can enhance polysaccharide-specific antibody responses. The present studies demonstrate significant adjuvant activity of CpG ODN for antibody responses against Streptococcus pneumoniae polysaccharide types 19F and 6B induced by conjugates of 19F and 6B with the protein carrier CRM(197). BALB/c ByJ mice were injected with 19F-CRM(197) or 6B-CRM(197) with or without CpG ODN, and sera were tested for anti-19F or anti-6B antibodies by enzyme-linked immunosorbent assay. The polysaccharide-specific antibody response to 19F-CRM(197) alone was predominantly of the immunoglobulin G1 (IgG1) and IgM isotypes, but addition of CpG ODN markedly increased geometric mean titers of total anti-19F antibody (23-fold), anti-19F IgG2a (26-fold), and anti-19F IgG3 (>246-fold). The polysaccharide-specific antibody response to 6B-CRM(197) alone consisted only of IgM, but addition of CpG ODN induced high titers of anti-6B IgG1 (>78-fold increase), anti-6B IgG2a (>54-fold increase), and anti-6B IgG3 (>3,162-fold increase). CpG ODN also increased anti-CRM(197) IgG2a and IgG3. Adjuvant effects were not observed with control non-CpG ODN. Thus, CpG ODN significantly enhance antipolysaccharide IgG responses (especially IgG2a and IgG3) induced by these glycoconjugate vaccines.