Immunobiological Properties of Antigenic Preparations Streptococcus pneumoniae and their Mixtures

Relevance. Type-specific immunity does not protect against infection with other pneumococcal serotypes. The phenomenon of the change of serotypes dominating the population of Streptococcus pneumoniae is known, in part due to the intensive recombination process and the phenomenon of «capsule switching». Therefore, the development of a serotype-independent pneumococcal vaccine is an important global public health priority. Ams. Investigation of immunobiological properties of candidate components of a future vaccine with serotype-independent activity. Materials and methods. For immunization of mice, preparations of the capsular polysaccharide of pneumococcus serotype 3 (CPS) were used; protein-containing fraction (PCF) obtained from an aqueous extract of S. pneumoniae 6B cells; recombinant pneumolysin (Ply); mixtures of drugs (CPS + Plу; CPS + PCF; PCF + Plу); conjugate vaccine Prevnar 13 (manufactured by PFIZER Inc. USA). Mice were immunized intraperitoneally, 2 times with an interval of 14 days. Intact mice were used as a control group. To assess the humoral immune IgG response, the method of solid-phase ELISA was used. Phagocytic activity was studied at 7, 14, 21 and 28 days after the second immunization. The cytokine level was determined in the blood sera of mice after the second immunization 2, 4, 8, and 24 hours later on a NovoCyte flow cytometer (ACEA Biosciences, USA) using the MACSPIex CytoKine 10 Kit mouse (Miltenyi Biotec Inc., USA) according to the manufacturer's instructions. Results. Immunization of mice with Ply as well as mixtures with CPS and PCF caused a significant increase in the level of antibodies to Ply. It was found that there was no apparent decrease in the level of antigen-specific antibodies when antigens were administered in combination with others. Pneumolysin, used alone or in combination with PCF and CPS, induces the production of antiinflammatory cytokines IL-4, IL-10, and IL-5 detected throughout the study. This is confirmed by a study of the opsonophagocytic activity of neutrophils from immunized CPS + Ply, Ply + PCF and Ply mice; a significant increase in the number of eosinophils is observed in their blood due to the stimulation of their production of IL-5. Conclusions. As a result of the studies, it was shown that Ply, used alone or in combination with CPS and PCF, has the highest immunogenicity: it stimulates a significant increase in the level of specific antibodies, stimulates Th-2, and induces the production of anti-inflammatory cytokines.

Epidemiology of Streptococcus pneumoniae serotypes in children before and after pneumococcal vaccination

Aim. To investigate how the pneumococcal vaccination affects the distribution of Streptococcus pneumoniae serotypes.Materials and Methods. In 2011-2019, 1,852 healthy children (1,354 aged ≤ 5 years and 480 aged from 6 to 17 years) were examined for the nasopharyngeal pneumococcal carriage. Of them, 539 children were tested before the start of pneumococcal vaccination (2011-2014), while 1,313 were tested during the vaccine campaign (2015-2019). Pneumococcal strains were serotyped using multiplex polymerase chain reaction.Results. Streptococcus pneumoniae serotype distribution considerably differed between children ≤ 5 and 6-17 years of age. Serotypes 23F, 19F, 19A, 6AB, and 15BC were prevalent in children ≤ 5 years of age while the older children were characterised by a high prevalence of capsular serotypes (3 and 33AF/37), serogroup 9 (9AV and 9LN), non-typeable streptococci, as well as 19F, 6AB and 6CD serotypes. Vaccination was associated with a significantly decreased prevalence of Streptococcus pneumoniae carriage (from 41.5% to 19.2%) among children ≤ 5 years of age, while this reduction was less pronounced (from 13.5 to 9.0%) in older children. Vaccination led to the shift in the distribution of pneumococcal serotypes towards an increased prevalence of non-vaccine serotypes that was particularly prominent in children ≤ 5 years of age. In particular, vaccination reduced the prevalence of 23F and 19A pneumococcal serotypes but heightened prevalence of 11AD serotype and to the appearance of previously undetected serotypes such as 8, 10A, 17F, 22F, 24ABF, 34, and 39.Conclusion. Pneumococcal vaccination decreased prevalence of pneumococcal carriage, yet causing a serotype replacement effect requiring improved microbiological monitoring in children of all age groups.

Review of global use of licensed vaccines and development of new vaccines for the prevention of pneumococcal infection

Streptococcus pneumoniae infection is the most common cause of high morbidity and mortality among children under 5 years of age, immunocompromised people, and the elderly. Despite significant success, the approved pneumococcal conjugate and polysaccharide vaccines are of limited efficacy, providing protection against a small fraction of the known pneumococcal serotypes. The rapid spread of multidrug-resistant strains exacerbates the global challenge of treating infection caused by S. pneumoniae. At the same time, the emerging new strains dictate the need to include new serotypes into vaccines. In view of this, further improvement of vaccines for the prevention of pneumococcal infections is an urgent task. The aim of this study was to review advances in the development of polysaccharide, conjugate, whole-cell pneumococcal vaccines, as well as vaccines based on protein antigens and vaccines with an antigen delivery system. Genomics and proteomics data have helped to improve approaches to the creation of polysaccharide and protein-based vaccines, as well as whole-cell vaccines with the potential for population prophylactic coverage against various pneumococcal serotypes that are not included in the licensed pneumococcal vaccines. The method of antigen delivery to the cell is of great importance in the development of vaccines. The most promising strategy for improving pneumococcal vaccines is the creation of vaccines based on bacterium-like or synthetic particles carrying several antigens, including pneumococcal surface proteins. In conclusion, it should be noted that top-priority vaccines are those that provide a wide range of protection against circulating pneumococcal serotypes and, in addition to eliciting a systemic immune response, also induce local immunity.

Pneumococcal Serotype Identification by Capsular Sequence Typing (CST): A Modified Novel Approach for Serotyping Directly in Clinical Samples

As almost 60–70% of Invasive Pneumococcal Disease (IPD) is identified by nonculture methods in Greece, serotyping is of high importance for the better monitoring of pneumococcal serotypes due to the availability of conjugate vaccines. The aim of the study was the modification and direct application of the Capsular Sequence Typing (CST) assay in clinical samples in order to serotype Streptococcus pneumoniae culture-negative, Polymerase Chain Reaction (PCR_-positive samples, followed by CST group specific single-tube PCR assays. A two-step PCR modified assay was applied on a total of 306 samples (such as CSF, blood, pleural and middle ear fluids, isolates) obtained from 283 patients with IPD. The overall performance permits a rapid, accurate and cost-effective method for nonculture pneumococcal serotyping. As the management of IPD is closely related to the continuous monitoring of pneumococcal serotypes, the proposed approach proved to be a valuable tool for the typing and epidemiological monitoring of S. pneumoniae, for the evaluation of the overall impact of vaccination programs in the era of pneumococcal conjugate vaccines, in order to initiate the appropriate vaccination strategy.

Pneumococcal serotypes causing non-invasive pneumonia in adults from a South Indian tertiary care hospital and the impact of the newer conjugate vaccines

Background. Streptococcus pneumoniae is the leading cause of community-acquired pneumonia (CAP) in adults. Ageing, chronic conditions and comorbidities are important risk factors for pneumococcal pneumonia. Purpose. There is lack of data on the pneumococcal serotypes causing non-invasive pneumonia in India. This study aims to determine the prevalent pneumococcal serotypes causing non-invasive pneumonia, the associated comorbidities, and the coverage of both the available pneumococcal vaccines in India and conjugate vaccines that are currently undergoing clinical trials. Methods. A total of 280 subjects (aged >16 years) who had clinical symptoms correlating with radiological findings for non-invasive bacteremic pneumonia and microbiological evidence of S. pneumoniae between 2018 and 2020 were included. The clinical, demographic, radiological and microbiological findings were retrieved from the Hospital Information System (HIS). Results. The common serotypes in order of prevalence were 19F, 9V, 23F, 6B, 11A, 13, 34, 10A, 19A and 6A. The predominant non-vaccine serotypes were 13, 34, 35B, 31 and 16F. The associated radiological findings were pneumonic consolidation and multi-lobar involvement. Other coinfected bacterial pathogens included H. influenzae, S. aureus, K. pneumoniae and P. aeruginosa. Conclusion. The pneumococcal vaccines: PCV10/GSK, PCV10/SII, PCV13, PCV15, PCV20 and PPSV23 provide an overall serotype coverage of 36, 41, 47, 48, 61 and 69 %, respectively of S. pneumoniae causing non-invasive pneumonia in South India. Increasing catch-up vaccination using PCV10(SII) in pre-school children could have a more significant impact on reducing pneumococcal pneumonia in adults (>50 years) in terms of increased herd immunity at an affordable cost.

It Takes Two to Tango: Combining Conventional Culture with Molecular Diagnostics Enhances Accuracy of Streptococcus pneumoniae Detection and Pneumococcal Serogroup/Serotype determination in Carriage

Background: The specificity of molecular methods for the detection of Streptococcus pneumoniae carriage is under debate. We propose a procedure that increases the accuracy of molecular detection of live pneumococci in polymicrobial respiratory samples. Methods: Culture and qPCR methods were applied to detect S. pneumoniae and pneumococcal serotypes in 1549 nasopharyngeal samples collected in the Netherlands (n=972) and England (n=577) from 946 toddlers and 603 adults, and in paired oropharyngeal samples collected exclusively from 319 Dutch adults. Samples with no live pneumococci isolated at primary diagnostic culture yet generating pneumococcus-specific signal in qPCRs were re-examined with a second, qPCR-guided culture. Optimal Cq cut-offs for positivity in qPCRs were determined via receiver operating characteristic (ROC) curve analysis using isolation of live pneumococci from the primary and qPCR-guided cultures as reference. Results: Detection of S. pneumoniae and pneumococcal serotypes with qPCRs in cultured (culture-enriched) nasopharyngeal samples exhibited near-perfect agreement with conventional culture (Cohens kappa: 0.95). Molecular methods also displayed increased sensitivity of detection for multiple serotype carriage. Among paired samples from adults, the sensitivity of S. pneumoniae detection in primary nasopharyngeal plus oropharyngeal cultures was significantly lower compared with molecular detection in both culture-enriched samples together (p<0.0001) and also in culture-enriched oropharyngeal samples alone (p<0.05). Conclusions: The sensitivity of S. pneumoniae carriage surveillance can be greatly improved by complementing conventional culture with qPCR and vice versa. The specificity of molecular methods for the detection of live pneumococci can be enhanced by incorporating statistical procedures based on ROC curve analysis. The procedure we propose improves detection of S. pneumoniae carriage in adults in particular and enhances specificity of serotype carriage detection.

Extracellular Vesicles from Different Pneumococcal Serotypes Are Internalized by Macrophages and Induce Host Immune Responses

Bacterial extracellular vesicles are membranous ultrastructures released from the cell surface. They play important roles in the interaction between the host and the bacteria. In this work, we show how extracellular vesicles produced by four different serotypes of the important human pathogen, Streptococcus pneumoniae, are internalized by murine J774A.1 macrophages via fusion with the membrane of the host cells. We also evaluated the capacity of pneumococcal extracellular vesicles to elicit an immune response by macrophages. Macrophages treated with the vesicles underwent a serotype-dependent transient loss of viability, which was further reverted. The vesicles induced the production of proinflammatory cytokines, which was higher for serotype 1 and serotype 8-derived vesicles. These results demonstrate the biological activity of extracellular vesicles of clinically important pneumococcal serotypes.

Streptococcus pneumoniae serotypes that frequently colonise the human nasopharynx are common recipients of penicillin-binding protein gene fragments from Streptococcus mitis

Streptococcus pneumoniae is an important global pathogen that causes bacterial pneumonia, sepsis and meningitis. Beta-lactam antibiotics are the first-line treatment for pneumococcal disease, however, their effectiveness is hampered by beta-lactam resistance facilitated by horizontal genetic transfer (HGT) with closely related species. Although interspecies HGT is known to occur among the species of the genus Streptococcus , the rates and effects of HGT between Streptococcus pneumoniae and its close relatives involving the penicillin binding protein (pbp) genes remain poorly understood. Here we applied the fastGEAR tool to investigate interspecies HGT in pbp genes using a global collection of whole-genome sequences of Streptococcus mitis , Streptococcus oralis and S. pneumoniae . With these data, we established that pneumococcal serotypes 6A, 13, 14, 16F, 19A, 19F, 23F and 35B were the highest-ranking serotypes with acquired pbp fragments. S. mitis was a more frequent pneumococcal donor of pbp fragments and a source of higher pbp nucleotide diversity when compared with S. oralis . Pneumococci that acquired pbp fragments were associated with a higher minimum inhibitory concentration (MIC) for penicillin compared with pneumococci without acquired fragments. Together these data indicate that S. mitis contributes to reduced β-lactam susceptibility among commonly carried pneumococcal serotypes that are associated with long carriage duration and high recombination frequencies. As pneumococcal vaccine programmes mature, placing increasing pressure on the pneumococcal population structure, it will be important to monitor the influence of antimicrobial resistance HGT from commensal streptococci such as S. mitis .

Nonpneumococcal Strains Recently Recovered from Carriage Specimens and Expressing Capsular Serotypes Highly Related or Identical to Pneumococcal Serotypes 2, 4, 9A, 13, and 23A

ABSTRACT The polysaccharide capsule is a key virulence factor of Streptococcus pneumoniae. There are numerous epidemiologically important pneumococcal capsular serotypes, and recent findings have demonstrated that several of them are commonly found among nonpathogenic commensal species. Here, we describe 9 nonpneumococcal strains carrying close homologs of pneumococcal capsular biosynthetic (cps) loci that were discovered during recent pneumococcal carriage studies of adults in the United States and Kenya. Two distinct Streptococcus infantis strains cross-reactive with pneumococcal serotype 4 and carrying cps4-like capsular biosynthetic (cps) loci were recovered. Opsonophagocytic killing assays employing rabbit antisera raised against S. infantis US67cps4 revealed serotype 4-specific killing of both pneumococcal and nonpneumococcal strains. An S. infantis strain and two Streptococcus oralis strains, all carrying cps9A-like loci, were cross-reactive with pneumococcal serogroup 9 strains in immunodiffusion assays. Antiserum raised against S. infantis US64cps9A specifically promoted killing of serotype 9A and 9V pneumococcal strains as well as S. oralis serotype 9A strains. Serotype-specific PCR of oropharyngeal specimens from a recent adult carriage study in the United States indicated that such nonpneumococcal strains were much more common in this population than serotype 4 and serogroup 9 pneumococci. We also describe S. oralis and S. infantis strains expressing serotypes identical or highly related to serotypes 2, 13, and 23A. This study has expanded the known overlap of pneumococcal capsular serotypes with related commensal species. The frequent occurrence of nonpneumococcal strains in the upper respiratory tract that share vaccine and nonvaccine capsular serotypes with pneumococci could affect population immunity to circulating pneumococcal strains. IMPORTANCE The distributions and frequencies of individual pneumococcal capsular serotypes among nonpneumococcal strains in the upper respiratory tract are unknown and potentially affect pneumococcal serotype distributions among the population and immunity to circulating pneumococcal strains. Repeated demonstration that these nonpneumococcal strains expressing so-called pneumococcal serotypes are readily recovered from current carriage specimens is likely to be relevant to pneumococcal epidemiology, niche biology, and even to potential strategies of employing commensal live vaccines. Here, we describe multiple distinct nonpneumococcal counterparts for each of the pneumococcal conjugate vaccine (PCV) serotypes 4 and 9V. Additional data from contemporary commensal isolates expressing serotypes 2, 13, and 23A further demonstrate the ubiquity of such strains. Increased focus upon this serological overlap between S. pneumoniae and its close relatives may eventually prove that most, or possibly all, pneumococcal serotypes have counterparts expressed by the common upper respiratory tract commensal species Streptococcus mitis, Streptococcus oralis, and Streptococcus infantis.

Carriage Dynamics of Pneumococcal Serotypes in Naturally Colonized Infants in a Rural African Setting During the First Year of Life

Streptococcus pneumoniae (the pneumococcus) carriage precedes invasive disease and influences population-wide strain dynamics, but limited data exist on temporal carriage patterns of serotypes due to the prohibitive costs of longitudinal studies. Here, we report carriage prevalence, clearance and acquisition rates of pneumococcal serotypes sampled from newborn infants bi-weekly from weeks 1 to 27, and then bi-monthly from weeks 35 to 52 in the Gambia. We used sweep latex agglutination and whole genome sequencing to serotype the isolates. We show rapid pneumococcal acquisition with nearly 31% of the infants colonized by the end of first week after birth and quickly exceeding 95% after 2 months. Co-colonization with multiple serotypes was consistently observed in over 40% of the infants at each sampling point during the first year of life. Overall, the mean acquisition time and carriage duration regardless of serotype was 38 and 24 days, respectively, but varied considerably between serotypes comparable to observations from other regions. Our data will inform disease prevention and control measures including providing baseline data for parameterising infectious disease mathematical models including those assessing the impact of clinical interventions such as pneumococcal conjugate vaccines.