Construction and Immunological Characterization of a Novel Nontoxic Protective Pneumolysin Mutant for Use in Future Pneumococcal Vaccines

Kirkham, Lea‐Ann S.; Kerr, A.; Douce, Gill; Paterson, Gavin K.; Dilts, Deborah A.; Liu, Dai-Fang; Mitchell, Tim

doi:10.1128/iai.74.1.586-593.2006

Cited by 98 publications

(90 citation statements)

References 49 publications

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“…Recombinant Ply and the pneumolysoid, delta6Ply, attenuated in respect of poreforming activity, were prepared as described previously [20,21]. The possible influence of contaminating endotoxin was excluded in both Ply preparations using the Endosafe ® -PTS TM system (Charles River Laboratories, Wilmington, MA, USA) which is based on the Limulus amebocyte lysate kinetic chromogenic method.…”

Section: Methodsmentioning

confidence: 99%

Pneumolysin Mediates Platelet Activation In Vitro

Nel

Durandt

Mitchell

et al. 2016

Lung

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This study has explored the role of the pneumococcal toxin, pneumolysin (Ply), in activating human platelets. Following exposure to Ply [10-80 nanograms (ng)/ml], platelet activation and cytosolic Ca 2+ concentrations were measured flow cytometrically according to the level of expression of CD62P (P-selectin) and spectrofluorimetrically respectively. Exposure to Ply resulted in marked upregulation of expression of platelet CD62P, achieving statistical significance at concentrations of 40 ng/ml and higher (p<0.05), in the setting of increased influx of Ca 2+ . These potentially pro-thrombotic actions of Ply were attenuated by depletion of Ca 2+ from the extracellular medium, or by exposure of the cells to a pneumolysoid devoid of pore-forming activity. These findings are consistent with a mechanism of Plymediated platelet activation involving sub-lytic pore formation, Ca 2+ influx, and mobilization of CD62P-expressing α-granules, which, if operative in vivo, may contribute to the pathogenesis of associated acute lung and myocardial injury during invasive pneumococcal disease.

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Section: Methodsmentioning

confidence: 99%

Pneumolysin Mediates Platelet Activation In Vitro

Nel

Durandt

Mitchell

et al. 2016

Lung

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“…10 The purities of both toxins were confirmed by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The stock protein concentrations of WT/PLY and Δ6 PLY were 493 and 768 mg/ml respectively and were essentially free of contaminating endotoxin (0.103 and 0.073 IU/mg).…”

Section: Methodsmentioning

confidence: 95%

“…Taken together with retention of immunogenicity and lack of toxicity in a murine model of pulmonary damage, 10 …”

Section: Discussionmentioning

confidence: 99%

“…One of the most promising of these is Δ6 PLY, generated by site-directed mutagenesis (deletion of alanine 146 and arginine 147 in the pore-forming region of wild-type PLY). 10 Critical criteria for further development include immunological and histological evaluation of protective efficacy and safety in animal models, as well as in vitro determination of attenuation of pore-forming activity in haemolytic assays. This latter assessment, although well-accepted, does not, however, detect the sub-lytic activity which underpins the potentially harmful pro-inflammatory activity of the toxin with other cell-types such as neutrophils 11 and epithelial cells.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of the interactions of the pneumolysoid, Δ6 PLY, with human neutrophils in vitro

Cockeran

Steel

Theron

et al. 2011

Vaccine

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The pneumolysin toxoid, Δ6 PLY, is a prototype pneumococcal protein vaccine candidate. However, its potentially detrimental residual proinflammatory interactions with human neutrophils are unknown. In the current study the effects of the toxoid (8-1000 ng/ml) have been compared with those of wild-type pneumolysin (WT/PLY, 8 ng/ml) on neutrophil cytosolic Ca 2+ fluxes, generation of leukotriene B 4 (LTB 4 ), and release of matrix metalloproteinase-9 (MMP-9), using spectrofluorimetric, and ELISA procedures (LTB 4 and MMP-9) respectively. Exposure of neutrophils to WT/PLY resulted in influx of Ca 2+ and significant (P<0.05) release of MMP-9 and generation of LTB 4 . However, treatment of the cells with Δ6 PLY at concentrations of up to 1000 ng/ml had only trivial effects on Ca 2+ influx and no effects on either release of MMP-9 or LTB 4 production. The observed absence of pro-inflammatory interactions of Δ6 PLY with neutrophils is clearly an important property of this pneumococcal protein vaccine candidate.

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“…Non‐haemolytic (detoxified) Ply (dPly) was generated from native Ply and provided by Professor T. Mitchell, at the time of generation affiliated with the Laboratory of Infection and Immunity, Glasgow Biomedical Research Centre, University of Glasgow, Scotland (currently at University of Birmingham, UK) 28. As described previously for other Ply alleles 29, Ply from serotype 1 ST306 was cloned into pET33b (Merck, Kenilworth, NJ, USA), and purified on a nickel column.…”

Section: Methodsmentioning

confidence: 99%

Cord blood Streptococcus pneumoniae-specific cellular immune responses predict early pneumococcal carriage in high-risk infants in Papua New Guinea

Francis

Richmond

Strickland

et al. 2016

Clinical and Experimental Immunology

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SummaryIn areas where Streptococcus pneumoniae is highly endemic, infants experience very early pneumococcal colonization of the upper respiratory tract, with carriage often persisting into adulthood. We aimed to explore whether newborns in high‐risk areas have pre‐existing pneumococcal‐specific cellular immune responses that may affect early pneumococcal acquisition. Cord blood mononuclear cells (CBMC) of 84 Papua New Guinean (PNG; high endemic) and 33 Australian (AUS; low endemic) newborns were stimulated in vitro with detoxified pneumolysin (dPly) or pneumococcal surface protein A (PspA; families 1 and 2) and compared for cytokine responses. Within the PNG cohort, associations between CBMC dPly and PspA‐induced responses and pneumococcal colonization within the first month of life were studied. Significantly higher PspA‐specific interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α, interleukin (IL)‐5, IL‐6, IL‐10 and IL‐13 responses, and lower dPly‐IL‐6 responses were produced in CBMC cultures of PNG compared to AUS newborns. Higher CBMC PspA‐IL‐5 and PspA‐IL‐13 responses correlated with a higher proportion of cord CD4 T cells, and higher dPly‐IL‐6 responses with a higher frequency of cord antigen‐presenting cells. In the PNG cohort, higher PspA‐specific IL‐5 and IL‐6 CBMC responses were associated independently and significantly with increased risk of earlier pneumococcal colonization, while a significant protective effect was found for higher PspA‐IL‐10 CBMC responses. Pneumococcus‐specific cellular immune responses differ between children born in pneumococcal high versus low endemic settings, which may contribute to the higher risk of infants in high endemic settings for early pneumococcal colonization, and hence disease.

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Construction and Immunological Characterization of a Novel Nontoxic Protective Pneumolysin Mutant for Use in Future Pneumococcal Vaccines

Cited by 98 publications

References 49 publications

Pneumolysin Mediates Platelet Activation In Vitro

Pneumolysin Mediates Platelet Activation In Vitro

Characterization of the interactions of the pneumolysoid, Δ6 PLY, with human neutrophils in vitro

Cord blood Streptococcus pneumoniae-specific cellular immune responses predict early pneumococcal carriage in high-risk infants in Papua New Guinea

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