Welcome to the ISPPD 2022 Meeting Calendar

Background

Vaccines that confer serotype-independent protection and with low production costs are required to reduce the burden of diseases caused by Streptococcus pneumoniae (SPN). The development of these improved vaccines requires the identification of protein-based candidates that elicit protective immune responses. PnuBioVax (PBV) is a pneumococcal multi-protein vaccine candidate that has recently shown safety and immunogenicity in a Phase I Trial. We aim to investigate whether PBV-specific antibody levels and B cell numbers correlate with protection against Experimental Human Pneumococcal Challenge (EHPC) in cohorts form the UK and Malawi.

Methods

Pre- and post-inoculation sera and nasal wash samples from healthy volunteers previously enrolled in EHPC studies (Liverpool: SPN6B, 15B, 3; Malawi: SPN6B) were used to measure anti-PBV systemic and mucosal antibody titers. PBV-IgG-secreting memory B cells were assessed by ELISPOTs in pre- and post-inoculation PBMCs (SPN6B). Functionality of anti-PBV antibodies was determined by standardized agglutination assays using sera from PBV-immunized rabbits.

Results

Preliminary data showed that systemic anti-PBV antibody titres and PBV IgG-secreting memory B cells were increased post-inoculation in those volunteers colonised with SPN6B (Liverpool). In addition, in those volunteers colonised, high anti-PBV antibody titres pre-inoculation correlated with early clearance of colonisation. An increased number of PBV IgG-secreting memory B cells at baseline was observed in those volunteers protected against experimental colonisation.

Conclusions

These data suggest that anti-PBV responses could provide a correlate of serotype independent immunity. Further analysis will provide data for at risk populations and other SPN serotypes.

Acknowledgements: This project is funded by a BactiVac Pump-Priming award.

Background

Previous studies have identified several pneumococcal gene mutations that disproportionally affect meningitis patients. Here we aim to confirm previously reported associations and identify additional genes linked to meningitis.

Methods

Non-synonymous mutations in previously reported candidate genes, including pbp1B, pspC, zmpB, and pde1, were determined by whole genome sequencing. The association between a candidate mutation and syndrome (meningitis vs. non-meningitis) was evaluated using a linear mixed-effects model (LMM), implemented in the FaST-LMM software, to control for population structure. All k-mers of length 15 were identified from draft genome assemblies. The k-mers present in >5% but <95% of isolates, representing common variants, were evaluated for association with meningitis using LMM.

Results

Of the 11,015 invasive pneumococcal isolates identified in 2016-2019, 763 (7%) were cultured from meningitis patients. Compared to non-meningitis isolates, meningitis isolates had a higher proportion of pbp1B641C (21% vs. 10%), pspC1360A (34% vs. 31%), and zmpB638A (5% vs. 2%) variants. After controlling for population structure, each of the three variants was significantly associated with meningitis (p<0.05). LMM analysis on 2,083,856 k-mers estimated a heritability (h²) of 0.08. K-mers that showed genome-wide significant association (p<1×10^-8) with meningitis were identified in genes encoding ABC transporter ATP-binding protein (H020_RS0107080), AraC family transcriptional regulator (H020_RS0107070), and bacteriocin-associated integral membrane family protein (H020_RS0100550), among others.

Conclusions

The pneumococcal genome explained a modest amount of variation in clinical manifestations (meningitis vs. non-meningitis), although a larger proportion of such variation could be due to host and host-pathogen interactions. Further depiction of underlying mechanisms could help better understand bacterial pathogenesis.

Background

Streptococcus pneumoniae, akin to multiple other invasive mucosal respiratory pathogens in humans, consistently produces a protease that cleaves the predominant antibody in the respiratory tract, IgA1. The protease is expressed on the bacterial surface, inhibits opsonophagocytosis (OPS) of the organism by IgA1 and facilitates its adherence to respiratory epithelial cells.

Methods

We expressed the full-length lytic protease and a nonfunctional single amino acid mutant and generated 4 murine monoclonal antibodies (mMAb IP01-4) that bound the protein and showed varying abilities to neutralize its proteolytic activity. Phagocytosis was performed with human and murine neutrophils and complement. Mice were infected intranasally.

Results

In vitro, neutralization proteolytic activity restored the ability of human capsule-specific IgA1 to effect complement-dependent OPS of S. pneumoniae. In vivo, mMab IP04 supported protection by human IgA1 of mice for survival after intranasal pneumococcal infection in vivo by both Fc- and F(ab’)₂- dependent mechanisms. Neutralization by mMAb’s IP02 and IP04 was mediated by inhibition of binding of IgA1 to the protease, binding that was localized around key metalloproteinase site. Neutralizing mMAb IP04 inhibited cleavage of the Fc portion of IgA1 from the bacterial surface. In summary, antibodies generated to pneumococcal IgA1 protease prevent cleavage of this human antibody subclass, facilitate IgA1-dependent OPS in vitro and protect against fatal mucosal infection in vivo.

Conclusions

IgA1 protease may serve as an independent vaccine candidate or as an adjunct to polysaccharide or other pneumocccal protein vaccines to limit primary mucosal infections with this prominent human pathogen.

Background

Monoclonal antibodies (mAbs) that boost the host immune system are attractive
therapeutic candidates for infectious diseases harboring antibiotic resistance or with
emerging non-vaccine serotypes. Recent studies have shown that specific point
mutations in the Fc domain of antibodies can enhance antibody clustering into
hexameric structures, required for a more efficient complement activation. This study
examines the efficacy of capsule-specific human mAbs as a therapeutic approach
against S. pneumoniae.

Methods

Hexamerization-enhancing mutations were introduced in anti-capsule monoclonal antibdies. The functionality of new engeneered antibodies was assessed by flow cytometry and bacteria counting. To test their potencial in vivo, bacteriemic pneumonia model was stablished using BALB/c mice.

Results

We show that serogroup-6 specific mAbs harboring the hexamer-enhancing E430G or
E345K point mutation potently increase complement activation and phagocytosis of
clinical isolates of this serogroup. Bacterial killing assays demonstrated the strong
potency of engineered antibodies to induce neutrophil-dependent killing of serotype-6B.
Moreover, we compared the protective capacity of wildtype antibody with its
counterpart E345K variant against a mouse model of bacteremic pneumonia. Passive
immunization with either antibody before intranasal challenge with serotype-6A
induced increased degree of protection against pneumococcal bacteremia when mice
were treated with the complement-enhancing E345K mutant.

Conclusions

This study represents a first systematic approach to design effective therapeutic
antibodies against S. pneumoniae with increased potency to activate the human
complement system.

Background

Identification of serotype-independent correlates of protection against S. pneumoniae (Spn) infection will accelerate development of vaccines with broad coverage. In mice, nasopharyngeal-resident memory IL-17A-secreting CD4⁺ T-cells mediate pneumococcal clearance and systemic IL-17A responses predict resistance to colonisation. We evaluated whether humoral and cellular responses to conserved pneumococcal proteins predict resistance to experimental colonisation.

Methods

A multiplex Luminex assay was used to measure serum IgG to 75 conserved pneumococcal antigens pre- and post-intranasal inoculation with Spn6B in 39 healthy adults. Baseline PBMCs from 60 volunteers were stimulated with killed pneumococcus or 70 antigens. Secreted concentrations of 4 cytokines/chemokines were correlated with subsequent pneumococcal carriage acquisition or density. PBMCs taken pre- and post-challenge were used to evaluate B-cell responses to five selected proteins by ELISpot.

Results

Baseline IgG or B-cell responses did not correlate with protection against colonisation. Successful colonisation associated with significantly increased antibody levels to five antigens and B-cell responses to two antigens increased 29 days post-challenge, whereas there were no significant increases in IgG or B-cell responses in non-colonised volunteers. PBMC-elicited protein-specific IL-17A responses were detected at baseline but did not correlate with carriage acquisition or density. Protein-specific MCP-1 levels at baseline were higher in non-colonised volunteers.

Conclusions

Baseline IgG, B-cell or PBMC-elicited IL-17A responses to single protein antigens did not predict resistance to experimental colonisation in this study. The association between MCP-1, a key chemokine for monocyte recruitment, and colonisation may suggest a possible correlate of protection. Further work is needed to assess whether protein-specific immune responses post-colonisation protect against re-colonisation.

Background

Streptococcus pneumoniae remains a leading cause of bacterial pneumonia. Despite widespread vaccination, a rise in infection and antibiotic resistance among nonvaccine serotypes has contributed to high disease incidence. Human monoclonal antibodies (mAbs) are gaining traction for the prevention and treatment of infectious diseases, and could provide an alternative and/or conjunctive option for pneumococcal disease treatment.

Methods

We isolated the first human mAbs to the pneumococcal histidine triad protein (PhtD). Binding analysis was completed by ELISA and biolayer interferometry. The serotype breadth of each mAb was determined using ELISAs and flow cytometry. mAbs were tested in several mouse challenge models, and we conducted several in vivo studies to determine the mechanism of pneumococcal mAb protection.

Results

mAbs PhtD3, PhtD6, PhtD7, and PhtD8 target diverse epitopes, which were correlated to serotype breadth. Prophylactic administration of mAbs PhtD3 and PhtD7 protected mice in intranasal and intravenous infection models with pneumococcal serotypes 3 and 4. mAb PhtD3 rescued mice when administered 24 hrs after infection, and a PhtD3/PhtD7 cocktail had enhanced protective efficacy. mAb PhtD3 efficacy was dependent on macrophages and complement, but not neutrophils, and mAb PhtD3 reduced the bacterial burden in mouse lungs and blood. Furthermore, mAb PhtD3 prolonged the survival of mice in an influenza/pneumococcal co-infection model.

Conclusions

We demonstrated that human mAbs to PhtD target diverse epitopes and protect against lethal pneumococcal infection. Ongoing studies include the isolation of human mAbs to additional protein antigens, optimizing mAb delivery, testing of additional mAb cocktails, and determining mAb efficacy in additional viral secondary infection models.

Background

Streptococcus pneumoniae (Spn) colonises respiratory epithelia but can also invade lung cells causing pneumonia.

Methods

We developed an ex vivo model with human airway epithelial (HAE) cells harvested from lung biopsies to study Spn colonisation and translocation. Flow-cytometry, confocal imaging and electron microscopy studies identified the epithelial lineage with signs of differentiation (beating cilia, mucus, and tight junctions). HAE cells were challenged with Spn wild-type TIGR4 (wtSpn) or its isogenic ΔcomC quorum sensing-deficient mutant.

Results

ΔcomC mutant colonised significantly less than wtSpn at 6 h post-inoculation but at significantly higher levels at 19 h and 30 h. Translocation correlated inversely with colonisation density. Transepithelial electric resistance (TEER) decreased after pneumococcal infection and correlated with increased translocation for both strains.

Confocal imaging illustrated colocalisation of intracellular Spn with both cilia and zonulin-1 and prominent microcolony formation with wtSpn but disintegration of microcolony structures over time with ΔcomC mutant. ΔcomC caused a more pronounced release of both zonulin-1 and lactate dehydrogenase into the supernatant at later time points than wtSpn, suggesting that cytotoxicity is likely not the mechanism leading to translocation. There was a density- and time-dependent increase of inflammatory cytokines from human HAE cells infected with ΔcomC compared with wtSpn, including increased levels of the NLRP3 inflammasome-related IL-18.

Conclusions

In conclusion, our experiments indicate that ComC system allows a higher organisational level of population structure resulting in microcolony formation, increased early colonisation and subsequent translocation. We propose that ComC inactivation unleashes a very different and possibly more virulent phenotype that merits further investigation.

Background

Persisters are a subpopulation of cells that are tolerant to lethal concentrations of antibiotics. This phenotypic variation is widespread among bacterial species and is involved in a variety of chronic and recurrent infections. Surprisingly, little to nothing is known about persistence in S. pneumoniae. The aim of this study was to make a first characterization of persistence in Streptococcus pneumoniae.

Methods

First, a valid long-living in vitro model was optimized using different strategies (comparison of growth media, addition of catalase or choline chloride) to prolong stationary phase survival by setting-up planktonic growth curves which would enable in vitro persistence assays. Second, time-kill curves were obtained by treating pneumococci with high concentrations of antibiotics (amoxicillin, cefuroxime, moxifloxacin and vancomycin) in different growth phases. Mathematical modeling of these killing dynamics was used to assess the presence of persisters since these would lead to biphasic patterns. To exclude resistance, persisters were re-grown and time-kill curves as well as antibiotic susceptibility was determined.

Results

The best condition to reach stable stationary phase survival up till 24 hours was by using Mueller-Hinton Broth supplemented with 5% lysed horse blood as liquid growth medium. Pneumococci treated with 100-fold MIC of the antibiotics resulted in time-kill curves showing a biphasic killing pattern, which is proof of presence of persisters. Resistance was excluded as similar time-kill curves were obtained from regrown persisters compared to the original strain and they remained susceptible to the antibiotics.

Conclusions

This research provides a first proof that antibiotic-tolerant persister cells are present in Streptococcus pneumoniae cultures.