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Contains fulltext : 291405.pdf (Publisher’s version ) (Open Access) INTRODUCTION: Several studies have shown that intradermal vaccination leads to improved immune responses. In addition, lowering vaccine doses will reduce costs and therefore potentially increase coverage. To determine whether intradermal delivery enhances the antibody responses against the 13-valent pneumococcal conjugate vaccine (PCV13), we compared intradermally and intramuscularly vaccinated mice. METHODS: Mice were immunized with PCV13, either intradermally or intramuscularly and CFU-counts in the nasal tissue were determined three or seven days after intranasal colonization with a serotype 4 clinical strain. Antibody concentrations against all thirteen polysaccharides were measured in blood and mucosal samples using a fluorescent-bead-based multiplex immunoassay. RESULTS: Antibody levels in both serum and mucosal samples were higher in the intramuscularly vaccinated group as compared to the intradermally vaccinated group. No protection against S. pneumoniae intranasal colonization was observed for either vaccination route. CONCLUSIONS: Intradermal vaccination was inferior to intramuscular immunization in inducing serotype-specific antibodies.

Background New hemocytometric parameters can be used to differentiate causes of acute febrile illness (AFI). We evaluated a software algorithm–Infection Manager System (IMS)—which uses hemocytometric data generated by Sysmex hematology analyzers, for its accuracy to detect bacteremia in AFI patients with and without malaria in Burkina Faso. Secondary aims included comparing the accuracy of IMS with C-reactive protein (CRP) and procalcitonin (PCT). Methods In a prospective observational study, patients of ≥ three-month-old (range 3 months– 90 years) presenting with AFI were enrolled. IMS, blood culture and malaria diagnostics were done upon inclusion and additional diagnostics on clinical indication. CRP, PCT, viral multiplex PCR on nasopharyngeal swabs and bacterial- and malaria PCR were batch-tested retrospectively. Diagnostic classification was done retrospectively using all available data except IMS, CRP and PCT results. Findings A diagnosis was affirmed in 549/914 (60.1%) patients and included malaria (n = 191) bacteremia (n = 69), viral infections (n = 145), and malaria-bacteremia co-infections (n = 47). The overall sensitivity, specificity, and negative predictive value (NPV) of IMS for detection of bacteremia in patients of ≥ 5 years were 97.0% (95% CI: 89.8–99.6), 68.2% (95% CI: 55.6–79.1) and 95.7% (95% CI: 85.5–99.5) respectively, compared to 93.9% (95% CI: 85.2–98.3), 39.4% (95% CI: 27.6–52.2), and 86.7% (95% CI: 69.3–96.2) for CRP at ≥20mg/L. The sensitivity, specificity and NPV of PCT at 0.5 ng/ml were lower at respectively 72.7% (95% CI: 60.4–83.0), 50.0% (95% CI: 37.4–62.6) and 64.7% (95% CI: 50.1–77.6) The diagnostic accuracy of IMS was lower among malaria cases and patients, Author summary This study describes the diagnostic accuracy of the Infection Manager System (IMS), a novel diagnostic algorithm for febrile illnesses that is equipped on a routine hematology analyzer. The latest generation hematology analyzers allow better differentiation between leukocyte subsets and their phenotype. The IMS was created, using differences in immune cell subsets (their activation status for instance), to differentiate viral from bacterial etiologies of fever. Such a tool may guide clinicians in their decision the initiate or withhold antimicrobial therapy. The study was carried out among febrile patients aged 3 months and older in rural Burkina Faso, a sub-Saharan African setting where malaria is endemic. Standard microbiological techniques such as blood culture were used as a reference to assess the diagnostic accuracy of IMS. We then compared the diagnostic accuracy of the IMS with the marketed biomarkers C-reactive protein (CRP) and procalcitonin (PCT). Our study showed that the diagnostic performance of the IMS was similar to CRP and better than PCT to detect bacteremia in patients with and without malaria co-infection. Further studies are needed to see if the IMS can be safely used to guide initial antimicrobial treatment and help to reduce further spread of antimicrobial resistance.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite for pneumococcal transmission and disease. Current vaccines protect only against disease and colonization caused by a limited number of serotypes, consequently allowing serotype replacement and transmission. Therefore, the development of a broadly protective vaccine against colonization, transmission and disease is desired but requires a better understanding of pneumococcal adaptation to its natural niche. Hence, we measured the levels of free and protein-bound transition metals in human nasal fluid, to determine the effect of metal concentrations on the growth and proteome of S. pneumoniae. Pneumococci cultured in medium containing metal levels comparable to nasal fluid showed a highly distinct proteomic profile compared to standard culture conditions, including the increased abundance of nine conserved, putative surface-exposed proteins. AliA, an oligopeptide binding protein, was identified as the strongest protective antigen, demonstrated by the significantly reduced bacterial load in a murine colonization and a lethal mouse pneumonia model, highlighting its potential as vaccine antigen.

Contains fulltext : 225116.pdf (Publisher’s version ) (Open Access) Bordetella pertussis vaccine escape mutants that lack expression of the pertussis antigen pertactin (Prn) have emerged in vaccinated populations in the last 10-20 years. Additionally, clinical isolates lacking another acellular pertussis (aP) vaccine component, filamentous hemagglutinin (FHA), have been found sporadically. Here, we show that both whole-cell pertussis (wP) and aP vaccines induced protection in the lungs of mice, but that the wP vaccine was more effective in nasal clearance. Importantly, bacterial populations isolated from the lungs shifted to an FHA-negative phenotype due to frameshift mutations in the fhaB gene. Loss of FHA expression was strongly selected for in Prn-deficient strains in the lungs following aP but not wP vaccination. The combined loss of Prn and FHA led to complete abrogation of bacterial surface binding by aP-induced serum antibodies. This study demonstrates vaccine- and anatomical site-dependent adaptation of B. pertussis and has major implications for the design of improved pertussis vaccines.

Contains fulltext : 177354.pdf (Publisher’s version ) (Open Access) Vaccination has been one of the most successful strategies to reduce morbidity and mortality caused by respiratory infections. Recent evidence suggests that differences in the host genetic background and environmental factors may contribute to heterogeneity in the immune response to vaccination. During pre-clinical testing, vaccines are often evaluated in a single mouse inbred strain, which may not translate well to the heterogeneous human population. Here, we examined the influence of host genetic background on vaccine-induced protection against pneumococcal colonization in two commonly used inbred mouse strains, i.e. C57BL/6 and BALB/cas well as the F1 cross of these two strains. Groups of mice were vaccinated intranasally with a vaccine formulation containing a model pneumococcal antigen, i.e. pneumococcal surface protein A (PspA), adjuvanted with cholera toxin subunit B (CTB). Even in the absence of vaccination, differences in colonization density were observed between mouse strains. Although vaccination significantly reduced pneumococcal density in all mouse strains, differences were observed in the magnitude of protection. We therefore examined immunological parameters known to be involved in vaccine-induced mucosal clearance of S. pneumoniae. We found that PspA-specific IgG levels in nasal tissue differed between mouse strains, but in all cases it correlated significantly with a reduction in colonization. Furthermore, increased mucosal IL17A, but not IFNgamma, IL10, or IL4, was found to be mouse strain specific. This suggests that the reduction of bacterial load may be accompanied by a Th17 response in all genetic backgrounds, although the cytokine dynamics may differ. Increased insight into the different immune mechanisms that affect pneumococcal carriage will contribute to development of future vaccines against S. pneumoniae.

Background: Different clinical manifestations of invasive pneumococcal disease (IPD) have thus far mainly been explained by patient characteristics. Here we studied the contribution of pneumococcal genetic variation to IPD phenotype. Methods: The index cohort consisted of 349 patients admitted to two Dutch hospitals between 2000-2011 with pneumococcal bacteraemia. We performed genome-wide association studies to identify pneumococcal lineages, genes and allelic variants associated with 23 clinical IPD phenotypes. The identified associations were validated in a nationwide (n=482) and a post-pneumococcal vaccination cohort (n=121). The contribution of confirmed pneumococcal genotypes to the clinical IPD phenotype, relative to known clinical predictors, was tested by regression analysis. Results: Among IPD patients, the presence of pneumococcal gene slaA was a nationwide confirmed independent predictor of meningitis (OR=10.5, p=0.001, 5% presence), as was sequence cluster 9 (predominant serotype 7F, OR=3.68, p=0.057, 11% presence). A set of 4 pneumococcal genes co-located on a prophage was a confirmed independent predictor of 30-day mortality (OR=3.4, p=0.003, 48% presence). We could detect the pneumococcal variants of concern in these patients' blood samples. Conclusions: In this study, knowledge of pneumococcal genotypic variants improved the clinical risk assessment for detrimental manifestations of IPD. This provides us with novel opportunities to target, anticipate or avert the pathogenic effects related to particular pneumococcal variants, and indicates that information on pneumococcal genotype is important for the diagnostic and treatment strategy in IPD. Ongoing surveillance is warranted to monitor the clinical value of information on pneumococcal variants in dynamic microbial and susceptible host populations.

Streptococcus pneumoniae is a major cause of life-threatening infections. Complement activation plays a vital role in opsonophagocytic killing of pneumococci in blood. Initial complement activation via the classical and lectin pathways is amplified through the alternative pathway amplification loop. Alternative pathway activity is inhibited by complement factor H (FH). Our study demonstrates the functional consequences of the variability in human serum FH levels on host defense. Using an in vivo mouse model combined with human in vitro assays, we show that the level of serum FH correlates with the efficacy of opsonophagocytic killing of pneumococci. In summary, we found that FH levels determine a delicate balance of alternative pathway activity, thus affecting the resistance to invasive pneumococcal disease. Our results suggest that variation in FH expression levels, naturally occurring in the human population, plays a thus far unrecognized role in the resistance to invasive pneumococcal disease.

Bacterial outer membrane vesicles (OMVs) are attractive vaccine formulations because they have intrinsic immunostimulatory properties. In principle, heterologous antigens incorporated into OMVs will elicit specific immune responses, especially if presented at the vesicle surface and thus optimally exposed to the immune system. In this study, we explored the feasibility of our recently developed autotransporter Hbp platform, designed to efficiently and simultaneously display multiple antigens at the surface of bacterial OMVs, for vaccine development. Using two Streptococcus pneumoniae proteins as model antigens, we showed that intranasally administered Salmonella OMVs displaying high levels of antigens at the surface induced strong protection in a murine model of pneumococcal colonization, without the need for a mucosal adjuvant. Importantly, reduction in bacterial recovery from the nasal cavity was correlated with local production of antigen-specific IL-17A. Furthermore, the protective efficacy and the production of antigen-specific IL-17A, and local and systemic IgGs, were all improved at increased concentrations of the displayed antigen. This discovery highlights the importance of an adequate antigen expression system for development of recombinant OMV vaccines. In conclusion, our findings demonstrate the suitability of the Hbp platform for development of a new generation of OMV vaccines, and illustrate the potential of using this approach to develop a broadly protective mucosal pneumococcal vaccine.

BackgroundDifferent clinical manifestations of invasive pneumococcal disease (IPD) have thus far mainly been explained by patient characteristics. Here we studied the contribution of pneumococcal genetic variation to IPD phenotype.MethodsThe index cohort consisted of 349 patients admitted to two Dutch hospitals between 2000-2011 with pneumococcal bacteraemia. We performed genome-wide association studies to identify pneumococcal lineages, genes and allelic variants associated with 23 clinical IPD phenotypes. The identified associations were validated in a nationwide (n=482) and a post-pneumococcal vaccination cohort (n=121). The contribution of confirmed pneumococcal genotypes to the clinical IPD phenotype, relative to known clinical predictors, was tested by regression analysis.FindingsThe presence of pneumococcal gene slaA was a nationwide confirmed independent predictor of meningitis (OR=10.5, p=0.001), as was sequence cluster 9 (OR=3.68, p=0.057). A set of 4 pneumococcal genes co-located on a prophage was a confirmed independent predictor of 30-day mortality (OR=3.4, p=0.003). We could detect the pneumococcal variants of concern in these patients’ blood samples by molecular amplification. In the post-vaccination cohort where the distribution of both patient characteristics and pneumococcal serotypes had changed, the relative importance of the prophage was no longer supported.InterpretationKnowledge of pneumococcal genotypic variants improved our clinical risk assessment for detrimental manifestations of IPD. This provides us with novel opportunities to target, anticipate or avert the pathogenic effects that are related to particular pneumococcal variants. Therefore, future diagnostics should facilitate prompt appreciation of pathogen diversity in clinical sepsis management. Ongoing surveillance is warranted to monitor the clinical value of information on pathogen variants in dynamic microbial and susceptible host populations.FundingNone.

Item does not contain fulltext For many bacterial respiratory infections, development of (severe) disease is preceded by asymptomatic colonization of the upper airways. For Streptococcus pneumoniae, the transition to severe lower respiratory tract infection is associated with an increase in nasopharyngeal colonization density. Insight into how the mucosal immune system restricts colonization may provide new strategies to prevent clinical symptoms. Several studies have provided indirect evidence that the mucosal adjuvant cholera toxin subunit B (CTB) may confer nonspecific protection against respiratory infections. Here, we show that CTB reduces the pneumococcal load in the nasopharynx, which required activation of the caspase-1/11 inflammasome, mucosal T cells, and macrophages. Our findings suggest that CTB-dependent activation of the local innate response synergizes with noncognate T cells to restrict bacterial load. Our study not only provides insight into the immunological components required for containment and clearance of pneumococcal carriage, but also highlights an important yet often understudied aspect of adjuvants.

Contains fulltext : 167230.PDF (Publisher’s version ) (Open Access) Streptococcus pneumoniae is a common cause of sepsis. Effective complement activation is an important component of host defence against invading pathogens, whilst excessive complement activation has been associated with endothelial dysfunction and organ damage. The alternative pathway amplification loop is important for the enhancement of complement activation. Factor H is a key negative regulator of the alternative pathway amplification loop and contributes to tight control of complement activation. We assessed the effect of inhibition of the alternative pathway on sepsis associated inflammation and disease severity using human factor H treatment in a clinically relevant mice model of pneumococcal sepsis. Mice were infected intravenously with live Streptococcus pneumoniae. At the first clinical signs of infection, 17 hours post-infection, mice were treated with ceftriaxone antibiotic. At the same time purified human factor H or in controls PBS was administered. Treatment with human factor H did not attenuate disease scores, serum pro-inflammatory cytokines, or vascular permeability and did not significantly affect C3 and C3a production at 26 h post-infection. Therefore, we conclude that inhibition of the alternative complement pathway by exogenous human factor H fails to attenuate inflammation and vascular leakage at a clinically relevant intervention time point in pneumococcal sepsis in mice.

Item does not contain fulltext Widespread vaccination against Streptococcus pneumoniae (the pneumococcus) has significantly reduced pneumococcal disease caused by vaccine serotypes. Despite vaccination, overall pneumococcal colonization rates in children have not reduced and otitis media (OM) by non-vaccine serotypes remains one of the most common childhood infections. Pneumococcal surface protein A (PspA) has been shown to be a promising protein antigen to induce broad protection against pneumococcal colonization. However, its ability to protect against OM remains unclear. Using our previously established mouse model of influenza-virus induced pneumococcal OM, we here show that intranasal vaccination of mice with PspA together with the mucosal adjuvant CTB results in a decrease in pneumococcal load in the middle ears. This decrease correlated with the induction of PspA-specific IgA, a balanced IgG1:IgG2a antibody response and the induction of a mucosal Th17 response. Our data suggests that the IL-17 response to PspA is more important for protection against OM, whilst the presence of antibodies may be less important, as determined in mice deficient in IL-17 signaling or antibody production. Together, these results suggest that mucosal vaccination with PspA may not only protect against colonization, but also against the development of virus-induced pneumococcal OM.

Contains fulltext : 154083.pdf (Publisher’s version ) (Open Access) Btg1 and Btg2 encode highly homologous proteins that are broadly expressed in different cell lineages, and have been implicated in different types of cancer. Btg1 and Btg2 have been shown to modulate the function of different transcriptional regulators, including Hox and Smad transcription factors. In this study, we examined the in vivo role of the mouse Btg1 and Btg2 genes in specifying the regional identity of the axial skeleton. Therefore, we examined the phenotype of Btg1 and Btg2 single knockout mice, as well as novel generated Btg1-/-;Btg2-/- double knockout mice, which were viable, but displayed a non-mendelian inheritance and smaller litter size. We observed both unique and overlapping phenotypes reminiscent of homeotic transformation along the anterior-posterior axis in the single and combined Btg1 and Btg2 knockout animals. Both Btg1-/- and Btg2-/- mice displayed partial posterior transformation of the seventh cervical vertebra, which was more pronounced in Btg1-/-;Btg2-/- mice, demonstrating that Btg1 and Btg2 act in synergy. Loss of Btg2, but not Btg1, was sufficient for complete posterior transformation of the thirteenth thoracic vertebra to the first lumbar vertebra. Moreover, Btg2-/- animals displayed complete posterior transformation of the sixth lumbar vertebra to the first sacral vertebra, which was only partially present at a low frequency in Btg1-/- mice. The Btg1-/-;Btg2-/- animals showed an even stronger phenotype, with L5 to S1 transformation. Together, these data show that both Btg1 and Btg2 are required for normal vertebral patterning of the axial skeleton, but each gene contributes differently in specifying the identity along the anterior-posterior axis of the skeleton.