Background

Recently we showed that a single nucleotide polymorphism (SNP) in the raffinose pathway regulatory gene rafR accounts for differences in the capacity of clonally-related pneumococcal strains to cause localised versus systemic infection. However, the underlying mechanism for these differences remain unclear.

Methods

6h post intranasal infection, a time-point were the wildtype and rafR-swapped strains are present in the lungs at similar levels, RNA was isolated from murine lungs. Host and pathogen transcriptomes were then simulatenously sequenced.

Results

The Dual RNA-seq data highlighted that this SNP extensively impacts both bacterial and host transcriptomes in infected lungs, affecting bacterial carbohydrate utilization and host inflammatory responses. A crucial role was predicted for differential neutrophil recruitment in the distinct virulence profiles of the infecting strains. Single cell analysis revealed that reduced expression of the RafR regulon, driven by the single rafR SNP, leads to massive recruitment of neutrophils and bacterial clearance in the lungs. Importantly, the observed disease outcomes were confirmed by in vivo neutrophil depletion showing that early detection of bacteria by the host in the lung environment is crucial for effective clearance.

Conclusions

Dual RNA-seq provides a powerful tool for understanding complex host-pathogen interactions, revealing how a single pneumococcal SNP drives differential disease outcomes.

Background

Streptococcus pneumoniae (Spn) colonizes the nasopharynx asymptomatically but is also a leading cause of community-acquired pneumonia, otitis media, bacteremia, sepsis, and meningitis. Spn pathogenesis studies have largely focused on the middle ear, airway, bloodstream, and central nervous system. Yet, increasing evidence indicates that during severe infections, Spn gains access to other vital organs.

Methods

Using dual species RNA-seq transcriptomics and mouse models of asymptomatic colonization and invasive disease, we characterized the pneumococcal (3 strains) and host transcriptomes during colonization and disease. This included the nasopharynx, lungs, and blood. Bacterial and host regulated pathways were validated using a panel of knock-out Spn strains and/or treatment of mice with relevant molecules.

Results

Spn differentially regulates genes in vivo in a site-specific manner, including upregulation of diverse scavenger pathways within the nasopharynx. Core genes highly expressed at all disease sites, and therefore targets for intervention, were identified, including pspA. Novel insights include that pneumococcal infection induces a Type I interferon response during pneumonia and invasive disease, but not colonization.

Conclusions

The pneumococcal transcriptome varies in anatomical site-specific manner and is vastly different during asymptomatic colonization versus disease. Host transcriptomes during invasive disease provide novel insights about potential therapeutic targets.

Background

The increase in resistance to antibiotics in the treatment of bacterial infections is a global problem. Streptococcus pneumoniae infection is a bacterial infection responsible for one-fifth of child mortality in Africa. The aim of this study was to formulate and evaluate immuno stimulatory complexes (ISCOM) from Carica papaya leaves.

Methods

The ISCOM was formed using the ethanol injection method and saponin source from Carica papaya leaves. Immunological assays of neutrophil adhesion, carbon clearance, hemagglutinating antibody titre and acute toxicity test were conducted using healthy laboratory mice.

Results

The results were compared to standard drug (Levamisole). Neutrophil adhesion showed fairly significant increase (21.26±0.00) as against the levamisole (21.09±0.00). The increase phagocytic index (0.089± 0.017) shows stimulation of reticuloendothelial system when compared to both controls (distilled water = 0.032 ± 0.001; levamisole = ± 0.071 ± 0.001). At 5000mg/kg body weight of administered ISCOM, no death was recorded.

Conclusions

It can be concluded from this study that Carica papaya leaves has immunomodulatory capacity and has significant saponin yield which can serve as a component of ISCOM formulation. Carica papaya based ISCOM promises be useful in the defense against Streptococcus pneumoniae infections.

Background

The incidence of invasive pneumococcal disease (IPD) caused by serotype 19A has increased particularly among elderly in Finland after 10-valent pneumococcal conjugate vaccine (PCV10) introduction into infant vaccination program in 2010. Thereafter the genomic epidemiology of 19A has changed among elderly and young children. The most common serotype 19A multilocus sequence types (STs) causing IPD in children and elderly in the pre-PCV10 (ST193 and ST482) and in children (ST994 and ST671-SLV) and elderly (ST199 and ST994) during PCV10 period were studied for sensitivity to opsonophagocytosis.

Methods

The sensitivity of serotype 19A isolates belonging to five different STs (3 isolates per ST) and the 19A reference strain (DS3519-97, ST199) to opsonophagocytosis was compared using the standardized opsonophagocytosis assay (OPA). Each isolate was analyzed twice using pooled sera (n=3) from children immunized with PCV10. IgG concentrations were measured with multiplex immunoassay.

Results

The sensitivity of 19A isolates to opsonophagocytosis varied by ST. ST994 isolates were the least sensitive and required 4-fold higher 19A and 19F antibody concentrations for 30 % killing compared to ST199 isolates, which were the most sensitive.

Conclusions

Vaccine-driven immune selection may have favored the emergence of OPA non-sensitive ST994 clone in both age groups.

Background

The long-term impact of PCV10 against invasive pneumococcal disease (IPD) was evaluated in Finland in 2010-2016. The incidence of IPD caused by vaccine-serotypes and 6A decreased. For 19A the reduction was not significant and incidence increased towards the end of follow-up in older children. We investigated reasons for waning protection against 19A.

Methods

We measured serum IgG concentration (multiplex immunoassay) and functional activity (opsonophagocytic killing assay, OPA) against cross-reacting pneumococcal capsular polysaccharides (PPS) 19F and 19A in samples collected at 13 (n=29-41), 24 (n=59-63) and 36 months (n=66-68) from children vaccinated with PCV10 using 2+1 schedule. To assess the contribution of 19F/A specific antibodies to binding and killing of 19A antigen/bacteria, eight sera were retested after adsorbing with 2.5 mg/ml 19A-PPS.

Results

Mean antibody concentrations to 19F and OPA titers to 19F and 19A decreased with age. Pre-adsorption with 19A-PPS resulted in average in 23% and 88% inhibition of antibody binding to 19F- and 19A-PPS, respectively, and resulted in decrease of 19A-OPA titer from 64-512 to <4.

Conclusions

PCV10 induced functional antibodies against serotypes 19F and 19A, but only a fraction of antibodies bound to epitopes common to both PPS. Protection against vaccine-related serotype 19A may not remain sufficient when antibody levels wane.

Background

The aim of this systematic review is to assess the effect of prophylactic use of antipyretics on immune response following conjugate pneumococcal immunization.

Methods

A systematic review of literature until June 2019 was conducted in electronic databases of Pubmed and Scopus. Randomized controlled trials were included in the study with no age limitation.

Results

Οut of 3956 citations retrieved, a total of 4 RCTs including 2775 children were included in the review, related to the PCV7 (n=1), PCV10 (n=2) and PCV13 (n=1) vaccines. The prophylactic administration of paracetamol after PCV13 caused a significant decrease in the immune response to pneumococcal serotypes 3,4,5,6B,23F and after PCV10 to serotypes 1,4,5,6B,9V,14,18C. The most significant effect was noted following the immediate administration of antipyretic comparing to administer 4-6 hours later. However, the antibody titers remained above protective levels in 88-100% of the children. No effect on the immunogenicity of the vaccines was detected in the PCV7 study, as well as after ibuprofen administration for all vaccines.

Conclusions

The prophylactic antipyretic administration seems to affect immune response to certain pneumococcal serotypes. More well-designed studies need to be conducted in order to evaluate the clinical significance of this effect.

Background

Pneumococcal colonization of the upper respiratory tract is a highly dynamic process involving bacteria binding to mucus, as well as bacterial cleavage of mucus components to evade mucociliary clearance. Increased binding of Spn to nasal glycoconjugates leads to entrapment in mucus and to more successful host elimination of Spn by mucociliary activity.

Methods

We used a solid-phase adherence assay with immobilized mucus of human and murine origin to screen for Spn surface factors and enzymes that modify host glycoconjugates to study Spn-mucus associations; strains with differential mucus binding were further tested in a murine model of colonization.

Results

Capsule (CPS)-, neuraminidase (NanA, NanB)-, and O-glycosidase (Eng)-deficient mutants showed increased mucus binding in vitro. The capsule- and neuraminidase-deficient mutants had a colonization defect in vivo, consistent with their role in mucus binding. In vitro, incubation with Spn removed sialic acid from bound mucus and this activity was diminished with the Spn_DnanAnanB [MOU1] [AH2] [AH3] mutant strain. Pre-treatment of mucus with neuraminidase reduced bacterial binding, confirming the role of sialic acid removal in mucus evasion.

Conclusions

Our study highlights the importance of sialic acid during Spn colonization in vitro and in vivo, and identifies Spn factors and enzymes necessary for mucus evasion.

Background

It has been widely documented that an organism’s circadian rhythm can have a detrimental impact on its susceptibility to bacterial challenge and severity of infection. Previous literature has recorded significant differences in the survival time of mice following intraperitoneal infection at different times along the circadian cycle in experimental models of sepsis caused by Streptococcus pneumoniae, however the exact mechanisms behind this phenomenon are unknown. Following recent work in our group suggesting that the spleen is the major organ underpinning pneumococcal infection, we hypothesised that circadian-controlled processes within the spleen are responsible for inducing the differential survival times documented in vivo

Methods

We utilised in vivo experimental mouse models with intravenous infection occurring at opposite ends of the circadian cycle. Further, we performed quantitative analysis of bacterial numbers within organs recovered from these mice by utilising confocal laser scanning microscopy and quantitative pathology imaging.

Results

Here, we show that splenic bacterial burden, but not liver or blood counts, correlate with subsequent survival time. Further, intracellular bacterial foci vary in number and size specifically within a subset of splenic macrophages known as marginal zone metallophilic macrophages.

Conclusions

The survival time appears to be dependent on differential bacterial replication abilities specifically within marginal zone macrophages.

Background

The tight junction (TJ) belt contributes to epithelial integrity during pneumococcal invasive disease. We investigated the effect of S. pneumoniae (Spn) invasion and translocation on TJ proteins and the role of Com, a quorum sensing system essential for the evolution of Spn.

Methods

A pseudostratified human airway epithelial tissue (HAE) model was developed with bronchial cells from donors. HAE cells were infected with TIGR4, or TIGR4ΔcomC, at different densities and incubated for up to 30 h. Pneumococci colonizing and those translocated to the basolateral side were quantified. Transepithelial electrical resistance (TEER), confocal/electron microscopy, ELISA for TJ proteins, and transcriptional analysis of genes encoding for ZO-1, claudin-4, and claudin-7, were performed.

Results

Infection with TIGR4 induced a dose-dependent decrease of TEER that correlated with invasion/translocation. Unexpectedly, a further decrease of TEER, with increased invasion, was induced by TIGR4ΔcomC compared to wt. Supernatant levels of ZO-1 and claudin-7 were increased when bacteria translocated, and were higher in HAE infected with TIGR4ΔcomC. Transcription of the ZO-1 gene was downregulated during infection with either strain, and further downregulated during the translocation stage.

Conclusions

S. pneumoniae invades, and translocates through, HAE cells by targeting TJ proteins. Knocking-down Com resulted in greater invasion and enhanced disturbance of the TJ.

Background

Streptococcus pneumoniae serotype-1 is the predominant cause of invasive pneumococcal disease in sub-Saharan Africa, but the mechanism behind its increased invasiveness is not well understood.

Methods

To identify factors that make key contributions to serotype-1 disease pathogenesis, we used a BALB/c model of pneumococcal pneumonia. Disease severity of infection with African serotype-1 (ST217 and ST3081) was compared to serotypes 2 (D39), 5, 6B and 7F.

Results

BALB/c mice are normally highly resistant to pneumococcal pneumonia with little to no bacterial dissemination from lungs into the bloodstream, resulting in 100% survival rates. However, when BALB/c mice were intranasally infected with serotype-1 ST217, 100% mortality was observed with high levels of bacteraemia within 24 hours. Mice challenged with all other serotypes survived.

We show that serotype-1 produces pneumolysin in large quantities, rapidly released due to high levels of bacterial autolysis. This released pneumolysin induces substantial levels of cellular cytotoxicity and breakdown of tight junctions between cells, allowing a route for rapid bacterial dissemination from the respiratory tract into blood; offering an explanation for increased serotype-1 invasiveness.

Conclusions

In conclusion, serotype-1 virulence was driven by rapid bacterial autolysis, which led to the release of large quantities of pneumolysin, enabling rapid bacterial dissemination into the bloodstream.

Background

Antibody avidity measures the strength of antibody-antigen binding, providing information on antibody quality. Usually avidity tests require large sample volumes, especially for multiple antigens, which is not suitable for paediatric studies. We investigated whether a bead-based avidity assay specific to pneumococcal polysaccharides could be developed using small volume samples.

Methods

Pneumococcal polysaccharides (1,3,4,5,6B,7F,9V,14,18C,19A,19F,23F) were modified with DMTMM (4-(4,6-dimethyl[1,3,5]triazin-2-yl)-4-methyl-morpholinium) and conjugated to carboxylated beads. 1M NaSCN was used to dissociate weak hydrogen bonds between antibody and antigen. The assay was validated on longitudinally collected sera from 6 pneumococcal conjugate vaccinated infants at 1,4,9,10,23,24 months of age. Avidity index (AI) was determined as the serum IgG titre that remained bound after NaSCN treatment compared to untreated serum.

Results

AIs against all 12 polysaccharides were obtained. In general, AIs increased following PCV immunisation. However, AIs were highly variable between children at each time-point. The lowest AIs were observed for serotype 1 and 3.

Conclusions

We have developed an assay that simultaneously measures antibody avidity for 12 pneumococcal polysaccharides in <5mL serum, and will now apply this assay to a cohort of PCV10- and PCV13-vaccinated children. This high-throughput assay may be a useful alternative measure of antibody quality compared to more technical assays such as multiplex-opsonophagocytosis.

Background

Despite widespread use of PCV13, pneumococcal carriage remains high among Gambian infants. We investigated the role of biomass smoke exposure and inflammation in modulating pneumococcal carriage in The Gambia.

Methods

Rural Gambian children (n=120) were followed up at regular intervals from birth to two years of age. All infants received PCV13. Pneumococcal carriage was determined by quantitative PCR and inflammation by measuring plasma alpha-1 glycoprotein (AGP). Smoke exposure was self-reported by the mothers. Adjusted random effects regression models were applied to investigate the relationships between pneumococcal carriage, smoke exposure, and inflammation.

Results

Exposure to biomass smoke was significantly associated with a nearly 3-fold increase in the odds of pneumococcal carriage (OR 2.9, 95% CI: 1.13 - 7.5) and, in independent models, a 1/3-log10 increase in pneumococcal load (Coefficient 0.35, 95% CI: 0.11 - 0.59), compared to non-exposure. Inflammation (AGP) was significantly associated with an increased pneumococcal load (Coefficient 0.22, 95% CI: 0.03 - 0.41) in a model unadjusted for smoke exposure. Mediation analysis suggests that there are age, inflammation and smoke exposure interactions that may modify the effects of smoke exposure on pneumococcal carriage.

Conclusions

Biomass smoke exposure may be an important environmental factor driving pneumococcal carriage and loads among PCV-vaccinated Gambian children.

Background

Streptococcus pneumoniae is a natural colonizer of the human respiratory tract, and a global priority pathogen comprised of over 90 different serotypes. Respiratory epithelial cells are among the first to encounter the organism, however, their role in orchestrating the response to infection is unknown.

Methods

We compared epithelial cell responses to serotype 4 (Tigr4), causing lethal symptomatic infection in mice, to a serotype 6B, which induces asymptomatic infection even at high inoculum.

Results

We found these interactions lead to divergent responses in either asymptomatic, host-limiting infection, or symptomatic, invasive pneumococcal infection. Our data show that 6B induces a distinct inflammatory profile in comparison to Tigr4, which requires KDM6B, a eukaryotic histone demethylase, and the cytokine IL-11. At the molecular level, we reveal that KDM6B demethylation of histone H3 remodels the promoter of IL-11 for expression. Finally, through chemical inhibition of KDM6B enzymatic activity, or exogenous addition of IL-11, the asymptomatic and symptomatic host response to pneumococcus can be interchanged.

Conclusions

Thus, we show for the first time that asymptomatic infection is driving a host response dependent on KDM6B and IL-11, and these factors are critical in modulating a divergent response to different pneumococcal serotypes.

Background

Conjugate vaccines successfully target specific serotypes but also lead to serotype replacement. Alternative strategies include bacterial proteins. Selection of protein antigens would benefit from knowledge of those most abundantly expressed during stages of human pneumococcal pathogenesis. Furthermore, vaccine protein candidates must also be expressed in mice for testing of candidates.

Methods

We designed a Nanostring codeset and analyzed 200 genes encoding for bacterial surface-exposed proteins. We evaluated transcriptomic expression in mouse colonization, pneumonia and sepsis models, as well as clinical CSF samples and human controlled infection.

Results

The 30 genes most highly expressed in each system (mouse model, human samples) were identified. There was excellent correlation between mouse colonization, pneumonia and blood specimens (R>0.85). Correlations between human colonization and meningitis were moderate (R=0.56), as were those between mouse and human transcriptomic profiles. We identified several genes highly expressed in all mouse and human conditions. Two in particular encoded for proteins that we show conferred protection against colonization and induced opsonic antibodies.

Conclusions

We present a novel approach to identify putative protective pneumococcal antigens, based on transcriptomic analyses of pneumococcal RNA harvested in different conditions. We identified two novel potential candidates, which also highlights the utility of studying pneumococcal gene expression from human samples.

Background

In the experimental human pneumococcal challenge (EHPC) model, healthy adults are intranasally inoculated with a pneumococcal challenge strain. A panproteome Streptococcus pneumoniae array, containing over 2,600 pneumococcal proteins, was used to screen systemic and mucosal antibodies from EHPC volunteers.

Methods

IgG and IgA responses were profiled in a cohort of 150 volunteers challenged with serotype 6B pneumococci, half of whom were susceptible to experimental colonization and the other half remained protected. Serum and nasal wash samples collected pre- and post-EHPC were probed on the S. pneumoniae panproteome microarray with simultaneous detection of IgA and IgG binding.

Results

Hundreds of pneumococcal proteins were reactive with serum and nasal wash IgG and IgA. IgA- and IgG-reactive proteins showed high levels of overlap. However, over 200 proteins were reactive only with IgG. Antibodies against numerous proteins significantly increased following challenge. Unsupervised clustering showed subject-specific stability of antibody profiles in serum, but independently grouped profiles in nasal wash samples collected before and after challenge. No specific profile differences were observed in pre-EHPC samples between susceptible and protected groups, but the response to EHPC showed unique antigen reactivity patterns.

Conclusions

A single encounter with pneumococci can elicit broad changes in mucosal and systemic antibody responses to pneumococcal proteins.

Background

Among Rochester NY children, a dramatic increase in nasopharyngeal colonization by non-vaccine serotypes 35B and 15A pneumococci occurred after introduction of PCV13. During years 2010-2013 serotype 35B infrequently caused acute otitis media compared to serotype 15A

Methods

We investigated differences in 14 virulence genes between 35B and 15A strains in-vitro and 9 host innate cytokine gene responses in the nasopharynx of children using RT-PCR, normalized with two housekeeping genes, as possible explanations for the difference in virulence.

Results

: Seven of 14 virulence genes were differentially regulated when comparing 35B and 15A strains. ComA, phtD and phtE were upregulated, ciaR, pcpA, nanA and CapD were downregulated. LuxS comD, spxB, spsA, lytA, lytB and Ply showed no difference. In the nasopharyngx, IL17 and IL23 production were >10 fold higher during 35B colonization compared to 15A colonization.

Conclusions

Low AOM case/carriage ratio of 35B from 2010-2013 in Rochester NY children was associated with differential regulation of known virulence factors and mucosal immunological responses.