The molecular basis of adjuvant activity of pneumolysin

QR Microbiology;QR180 Immunology

Dalziel, Catherine Ellen ; Mitchell, Timothy J.

University:University of Glasgow

Department:Institute of Infection Immunity and Inflammation

关键词: Pneumolysin, adjuvant, macrophage, TLR4, IL-1 beta, complement.;   

Others  :  http://theses.gla.ac.uk/5405/1/2014dalzielPhd.pdf

来源: University of Glasgow

【 摘 要 】

Streptococcus pneumoniae is a major human pathogen and causes a significantburden of disease in both developed and developing countries. Currently, twopneumococcal vaccines are available, a polysaccharide conjugate vaccine forchildren <2 years of age and an adult polysaccharide vaccine for ‘at risk’ groupssuch as the elderly and immunocompromised. Unfortunately, due to the vastvariation and highly recombinant nature of the pneumococcus vaccine escapethrough serotype replacement is significantly decreasing the efficacy ofpneumococcal vaccines globally. New cost-effective and protective pneumococcalvaccines are urgently required.Pneumolysin (PLY) is a 53Kd cholesterol-dependent cytolysin that is largelyconserved in all strains of Streptococcus pneumoniae, making it an ideal candidatefor inclusion in a broad spectrum vaccine. It has been shown that PLY is not only aprotective immunogen but also has potent adjuvant properties and stimulates bothIgG and IgA antibody responses to antigens genetically coupled to the toxin (Douceet al., 2010). Both systemic and mucosal responses are induced when PLY is usedas an adjuvant which may prevent colonization and therefore provide non-serotypespecific herd immunity to Streptococcus pneumoniae. The cytolytic activity ofPLY prevents its inclusion in a human vaccine; a non-lytic deletion mutant 76PLYwas created for this purpose which retains adjuvanticity, albeit slightly reduced.The aim of this study was to elucidate the mechanism(s) of PLY/Δ6PLYadjuvanticity, it will be essential to have a basic model of adjuvant activity beforePLY-based vaccines can be advanced to human clinical trials.This project used a combination of high-throughput methods such as protein pulldownsand gene expression profiling to examine the abilities of PLY, 76PLY and thetruncation mutants D123PLY and D4PLY to bind to and be internalized by host cellsand to differentially regulate gene expression. These studies highlighted specificand direct interactions between PLY variants and the host cytoskeleton that couldmediate antigen/PLY uptake; they also revealed a pattern of gene expression thatis similar to those of other adjuvants and could provide the basis for a model ofadjuvanticity.Finally, through the use of reporter cell lines and transgenic TLR4-/- BMDM, therelationship between PLY and TLR4 has been further defined. A novel method forpreparing vehicle controls provided evidence that the ligation of TLR4 in thissystem is PLY-dependent and is not an artefact caused by contaminating TLRligands such as LPS. Once this was established it was possible to furtherinvestigate the role of TLR4 in the adjuvant activity of PLY, in particular the PLYdependentproduction of IL-1@. Through these studies a surprising role for TLR4 inin vitro PLY-dependent cytokine production was discovered. Additionally, it wasfound that complement has an essential role in the PLY-dependent production ofIL-1@. The role(s) of complement and IL-1@ in the adjuvant activity were furtherinvestigated using an in vivo immunization model and the biological basis for thedifference in adjuvant activity of PLY and 76PLY was defined.