Changes in capsular serotype alter the surface exposure of pneumococcal adhesins and impact virulence

We examined the contribution of serotype on Streptococcus pneumoniae adhesion and virulence during respiratory tract infection using a panel of isogenic TIGR4 (serotype 4) mutants expressing the capsule types 6A (+6A), 7F (+7F) and 23F (+23F) as well as a deleted and restored serotype 4 (+4) control strain. Immunoblots, bacterial capture assays with immobilized antibody, and measurement of mean fluorescent intensity by flow cytometry following incubation of bacteria with antibody, all determined that the surface accessibility, but not total protein levels, of the virulence determinants Pneumococcal surface protein A (PspA), Choline binding protein A (CbpA), and Pneumococcal serine-rich repeat protein (PsrP) changed with serotype. In vitro, bacterial adhesion to Detroit 562 pharyngeal or A549 lung epithelial cells was modestly but significantly altered for +6A, +7F and +23F. In a mouse model of nasopharyngeal colonization, the number of +6A, +7F, and +23F pneumococci in the nasopharynx was reduced 10 to 100-fold versus +4; notably, only mice challenged with +4 developed bacteremia. Intratracheal challenge of mice confirmed that capsule switch strains were highly attenuated for virulence. Compared to +4, the +6A, +7F, and +23F strains were rapidly cleared from the lungs and were not detected in the blood. In mice challenged intraperitoneally, a marked reduction in bacterial blood titers was observed for those challenged with +6A and +7F versus +4 and +23F was undetectable. These findings show that serotype impacts the accessibility of surface adhesins and, in particular, affects virulence within the respiratory tract. They highlight the complex interplay between capsule and protein virulence determinants

Progress in mucosal immunization for protection against pneumococcal pneumonia

Introduction: Lower respiratory tract infections are the fourth cause of death worldwide and pneumococcus is the leading cause of pneumonia. Nonetheless, existing pneumococcal vaccines are less effective against pneumonia than invasive diseases and serotype replacement is a major concern. Protein antigens could induce serotype-independent protection, and mucosal immunization could offer local and systemic immune responses and induce protection against pneumococcal colonization and lung infection. Areas covered: Immunity induced in the experimental human pneumococcal carriage model, approaches to address the physiological barriers to mucosal immunization and improve delivery of the vaccine antigens, different strategies already tested for pneumococcal mucosal vaccination, including live recombinant bacteria, nanoparticles, bacterium-like particles, and nanogels as well as, nasal, pulmonary, sublingual and oral routes of vaccination. Expert opinion: The most promising delivery systems are based on nanoparticles, bacterial-like particles or nanogels, which possess greater immunogenicity than the antigen alone and are considered safer than approaches based on living cells or toxoids. These particles can protect the antigen from degradation, eliminating the refrigeration need during storage and allowing the manufacture of dry powder formulations. They can also increase antigen uptake, control release of antigen and trigger innate immune responses

Recombinant plants provide a new approach to the production of bacterial polysaccharide for vaccines

Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections

The Ocular Conjunctiva as a Mucosal Immunization Route: A Profile of the Immune Response to the Model Antigen Tetanus Toxoid

Background: In a quest for a needle-free vaccine administration strategy, we evaluated the ocular conjunctiva as an alternative mucosal immunization route by profiling and comparing the local and systemic immune responses to the subcutaneous or conjunctival administration of tetanus toxoid (TTd), a model antigen. Materials and methods: BALB/c and C57BL/6 mice were immunized either subcutaneously with TTd alone or via the conjunctiva with TTd alone, TTd mixed with 2% glycerol or TTd with merthiolate-inactivated whole-cell B. pertussis (wBP) as adjuvants. Mice were immunized on days 0, 7 and 14 via both routes, and an evaluation of the local and systemic immune responses was performed two weeks after the last immunization. Four weeks after the last immunization, the mice were challenged with a lethal dose (2 x LD50) of tetanus toxin. Results: The conjunctival application of TTd in BALB/c mice induced TTd-specific secretory IgA production and skewed the TTd-specific immune response toward a Th1/Th17 profile, as determined by the stimulation of IFN gamma and IL-17A secretion and/or the concurrent pronounced reduction of IL-4 secretion, irrespective of the adjuvant. In conjunctivaly immunized C57BL/6 mice, only TTd administered with wBP promoted the establishment of a mixed Th1/Th17 TTd-specific immune response, whereas TTd alone or TTd in conjunction with glycerol initiated a dominant Th1 response against TTd. Immunization via the conjunctiva with TTd plus wBP adjuvant resulted in a 33% survival rate of challenged mice compared to a 0% survival rate in non-immunized animals (p lt 0.05). Conclusion: Conjunctival immunization with TTd alone or with various adjuvants induced TTd-specific local and systemic immune responses, predominantly of the Th1 type. The strongest immune responses developed in mice that received TTd together with wBP, which implies that this alternative route might tailor the immune response to fight intracellular bacteria or viruses more effectively

Decrease in Pneumococcal Co-Colonization following Vaccination with the Seven-Valent Pneumococcal Conjugate Vaccine

Understanding the epidemiology of pneumococcal co-colonization is important for monitoring vaccine effectiveness and the occurrence of horizontal gene transfer between pneumococcal strains. In this study we aimed to evaluate the impact of the seven-valent pneumococcal conjugate vaccine (PCV7) on pneumococcal co-colonization among Portuguese children. Nasopharyngeal samples from children up to 6 years old yielding a pneumococcal culture were clustered into three groups: pre-vaccine era (n = 173), unvaccinated children of the vaccine era (n = 169), and fully vaccinated children (4 doses; n = 150). Co-colonization, serotype identification, and relative serotype abundance were detected by analysis of DNA of the total bacterial growth of the primary culture plate using the plyNCR-RFLP method and a molecular serotyping microarray-based strategy. The plyNCR-RFLP method detected an overall co-colonization rate of 20.1%. Microarray analysis confirmed the plyNCR-RFLP results. Vaccination status was the only factor found to be significantly associated with co-colonization: co-colonization rates were significantly lower (p = 0.004; Fisher's exact test) among fully vaccinated children (8.0%) than among children from the pre-PCV7 era (17.3%) or unvaccinated children of the PCV7 era (18.3%). In the PCV7 era there were significantly less non-vaccine type (NVT) co-colonization events than would be expected based on the NVT distribution observed in the pre-PCV7 era (p = 0.024). In conclusion, vaccination with PCV7 resulted in a lower co-colonization rate due to an asymmetric distribution between NVTs found in single and co-colonized samples. We propose that some NVTs prevalent in the PCV7 era are more competitive than others, hampering their co-existence in the same niche. This result may have important implications since a decrease in co-colonization events is expected to translate in decreased opportunities for horizontal gene transfer, hindering pneumococcal evolution events such as acquisition of antibiotic resistance determinants or capsular switch. This might represent a novel potential benefit of conjugate vaccines

Effect of Seven-Valent Pneumococcal Conjugate Vaccine on Staphylococcus aureus Colonisation in a Randomised Controlled Trial

Background: Heptavalent pneumococcal conjugate vaccine (PCV7) shifts nasopharyngeal colonisation with vaccine serotype pneumococci towards nonvaccine serotypes. Because of the reported negative association of vaccine serotype pneumococci and Staphylococcus aureus in the nasopharynx, we explored the effect of PCV7 on nasopharyngeal colonisation with S. aureus in children and parents. Methodology/Principal Findings: This study was part of a randomised controlled trial on the effect of PCV7 on pneumococcal carriage, enrolling healthy newborns who were randomly assigned (1: 1: 1) to receive PCV7 (1) at 2 and 4 months of age (2) at 2, 4 and 11 months or (3) no PCV7 (controls). Nasopharyngeal colonisation of S. aureus was a planned secondary outcome. Nasopharyngeal swabs were obtained from all children over a 2-year period with 6-months interval and from one parent at the child's age of 12 and 24 months and cultured for Streptococcus pneumoniae and S. aureus. Between July 2005 and February 2006, 1005 children were enrolled and received either 2-doses of PCV7 (n = 336), 2+1-doses (336) or no dose (n = 333) before PCV7 implementation in the Dutch national immunization program. S. aureus colonisation had doubled in children in the 2+1-dose group at 12 months of age compared with unvaccinated controls (10.1% versus 5.0%; p = 0.019). A negative association for co-colonisation of S. pneumoniae and S. aureus was observed for both vaccine serotype (adjusted odds ratio (aOR) 0.53, 95% confidence interval (CI) 0.38-0.74) and nonvaccine serotype pneumococci (aOR 0.67, 95% CI 0.52-0.88). Conclusions/Significance: PCV7 induces a temporary increase in S. aureus colonisation in children around 12 months of age after a 2+1-dose PCV7 schedule. The potential clinical consequences are unknown and monitoring is warranted

Cross-Protective Peptide Vaccine against Influenza A Viruses Developed in HLA-A*2402 Human Immunity Model

Background: The virus-specific cytotoxic T lymphocyte (CTL) induction is an important target for the development of a broadly protective human influenza vaccine, since most CTL epitopes are found on internal viral proteins and relatively conserved. In this study, the possibility of developing a strain/subtype-independent human influenza vaccine was explored by taking a bioinformatics approach to establish an immunogenic HLA-A24 restricted CTL epitope screening system in HLAtransgenic mice. Methodology/Principal Findings: HLA-A24 restricted CTL epitope peptides derived from internal proteins of the H5N1 highly pathogenic avian influenza A virus were predicted by CTL epitope peptide prediction programs. Of 35 predicted peptides, six peptides exhibited remarkable cytotoxic activity in vivo. More than half of the mice which were subcutaneously vaccinated with the three most immunogenic and highly conserved epitopes among three different influenza A virus subtypes (H1N1, H3N2 and H5N1) survived lethal influenza virus challenge during both effector and memory CTL phases. Furthermore, mice that were intranasally vaccinated with these peptides remained free of clinical signs after lethal virus challenge during the effector phase. Conclusions/Significance: This CTL epitope peptide selection system can be used as an effective tool for the development of a cross-protective human influenza vaccine. Furthermore this vaccine strategy can be applicable to the development o

Prevalence and Genetic Characterization of Pertactin-Deficient Bordetella pertussis in Japan

The adhesin pertactin (Prn) is one of the major virulence factors of Bordetella pertussis, the etiological agent of whooping cough. However, a significant prevalence of Prn-deficient (Prn−) B. pertussis was observed in Japan. The Prn− isolate was first discovered in 1997, and 33 (27%) Prn− isolates were identified among 121 B. pertussis isolates collected from 1990 to 2009. Sequence analysis revealed that all the Prn− isolates harbor exclusively the vaccine-type prn1 allele and that loss of Prn expression is caused by 2 different mutations: an 84-bp deletion of the prn signal sequence (prn1ΔSS, n = 24) and an IS481 insertion in prn1 (prn1::IS481, n = 9). The frequency of Prn− isolates, notably those harboring prn1ΔSS, significantly increased since the early 2000s, and Prn− isolates were subsequently found nationwide. Multilocus variable-number tandem repeat analysis (MLVA) revealed that 24 (73%) of 33 Prn− isolates belong to MLVA-186, and 6 and 3 Prn− isolates belong to MLVA-194 and MLVA-226, respectively. The 3 MLVA types are phylogenetically closely related, suggesting that the 2 Prn− clinical strains (harboring prn1ΔSS and prn1::IS481) have clonally expanded in Japan. Growth competition assays in vitro also demonstrated that Prn− isolates have a higher growth potential than the Prn+ back-mutants from which they were derived. Our observations suggested that human host factors (genetic factors and immune status) that select for Prn− strains have arisen and that Prn expression is not essential for fitness under these conditions

Identification of Candidate Susceptibility and Resistance Genes of Mice Infected with Streptococcus suis Type 2

Streptococcus suis type 2 (SS2) is an important swine pathogen and zoonosis agent. A/J mice are significantly more susceptible than C57BL/6 (B6) mice to SS2 infection, but the genetic basis is largely unknown. Here, alterations in gene expression in SS2 (strain HA9801)-infected mice were identified using Illumina mouse BeadChips. Microarray analysis revealed 3,692 genes differentially expressed in peritoneal macrophages between A/J and B6 mice due to SS2 infection. Between SS2-infected A/J and control A/J mice, 2646 genes were differentially expressed (1469 upregulated; 1177 downregulated). Between SS2-infected B6 and control B6 mice, 1449 genes were differentially expressed (778 upregulated; 671 downregulated). These genes were analyzed for significant Gene Ontology (GO) categories and signaling pathways using the Kyoto Encylopedia of Genes and Genomes (KEGG) database to generate a signaling network. Upregulated genes in A/J and B6 mice were related to response to bacteria, immune response, positive regulation of B cell receptor signaling pathway, type I interferon biosynthesis, defense and inflammatory responses. Additionally, upregulated genes in SS2-infected B6 mice were involved in antigen processing and presentation of exogenous peptides, peptide antigen stabilization, lymphocyte differentiation regulation, positive regulation of monocyte differentiation, antigen receptor-mediated signaling pathway and positive regulation of phagocytosis. Downregulated genes in SS2-infected B6 mice played roles in glycolysis, carbohydrate metabolic process, amino acid metabolism, behavior and muscle regulation. Microarray results were verified by quantitative real-time PCR (qRT-PCR) of 14 representative deregulated genes. Four genes differentially expressed between SS2-infected A/J and B6 mice, toll-like receptor 2 (Tlr2), tumor necrosis factor (Tnf), matrix metalloproteinase 9 (Mmp9) and pentraxin 3 (Ptx3), were previously implicated in the response to S. suis infection. This study identified candidate genes that may influence susceptibility or resistance to SS2 infection in A/J and B6 mice, providing further validation of these models and contributing to understanding of S. suis pathogenic mechanisms

Long-Term Effects of Pneumococcal Conjugate Vaccine on Nasopharyngeal Carriage of S. pneumoniae, S. aureus, H. influenzae and M. catarrhalis

BACKGROUND: Shifts in pneumococcal serotypes following introduction of 7-valent pneumococcal conjugate vaccine (PCV-7) may alter the presence of other bacterial pathogens co-inhabiting the same nasopharyngeal niche. METHODOLOGY/PRINCIPAL FINDINGS: Nasopharyngeal prevalence rates of S. pneumoniae, S. aureus, H. influenzae and M. catarrhalis were investigated before, 3 and 4.5 years after introduction of PCV-7 in the national immunisation program in children at 11 and 24 months of age, and parents of 24-month-old children (n≈330/group) using conventional culture methods. Despite a virtual disappearance of PCV-7 serotypes over time, similar overall pneumococcal rates were observed in all age groups, except for a significant reduction in the 11-month-old group (adjusted Odds Ratio after 4.5 years 0.48, 95% Confidence Interval 0.34-0.67). Before, 3 and 4.5 years after PCV-7 implementation, prevalence rates of S. aureus were 5%, 9% and 14% at 11 months of age (3.59, 1.90-6.79) and 20%, 32% and 34% in parents (1.96, 1.36-2.83), but remained similar at 24 months of age, respectively. Prevalence rates of H. influenzae were 46%, 65% and 65% at 11 months (2.22, 1.58-3.13), 52%, 73% and 76% at 24 months of age (2.68, 1.88-3.82) and 23%, 30% and 40% in parents (2.26, 1.58-3.33), respectively. No consistent changes in M. catarrhalis carriage rates were observed over time. CONCLUSIONS/SIGNIFICANCE: In addition to large shifts in pneumococcal serotypes, persistently higher nasopharyngeal prevalence rates of S. aureus and H. influenzae were observed among young children and their parents after PCV-7 implementation. These findings may have implications for disease incidence and antibiotic treatment in the post-PCV era