Streptococcus pneumoniae is a major human pathogen and causes a significant burden of disease in both developed and developing countries. Currently, two pneumococcal vaccines are available, a polysaccharide conjugate vaccine for children <2 years of age and an adult polysaccharide vaccine for ‘at risk’ groups such as the elderly and immunocompromised. Unfortunately, due to the vast variation and highly recombinant nature of the pneumococcus vaccine escape through serotype replacement is significantly decreasing the efficacy of pneumococcal vaccines globally. New cost-effective and protective pneumococcal vaccines are urgently required. Pneumolysin (PLY) is a 53Kd cholesterol-dependent cytolysin that is largely conserved in all strains of Streptococcus pneumoniae, making it an ideal candidate for inclusion in a broad spectrum vaccine. It has been shown that PLY is not only a protective immunogen but also has potent adjuvant properties and stimulates both IgG and IgA antibody responses to antigens genetically coupled to the toxin (Douce et al., 2010). Both systemic and mucosal responses are induced when PLY is used as an adjuvant which may prevent colonization and therefore provide non-serotype specific herd immunity to Streptococcus pneumoniae. The cytolytic activity of PLY prevents its inclusion in a human vaccine; a non-lytic deletion mutant 76PLY was created for this purpose which retains adjuvanticity, albeit slightly reduced. The aim of this study was to elucidate the mechanism(s) of PLY/Δ6PLY adjuvanticity, it will be essential to have a basic model of adjuvant activity before PLY-based vaccines can be advanced to human clinical trials. This project used a combination of high-throughput methods such as protein pulldowns and gene expression profiling to examine the abilities of PLY, 76PLY and the truncation mutants D123PLY and D4PLY to bind to and be internalized by host cells and to differentially regulate gene expression. These studies highlighted specific and direct interactions between PLY variants and the host cytoskeleton that could mediate antigen/PLY uptake; they also revealed a pattern of gene expression that is similar to those of other adjuvants and could provide the basis for a model of adjuvanticity. Finally, through the use of reporter cell lines and transgenic TLR4-/- BMDM, the relationship between PLY and TLR4 has been further defined. A novel method for preparing vehicle controls provided evidence that the ligation of TLR4 in this system is PLY-dependent and is not an artefact caused by contaminating TLR ligands such as LPS. Once this was established it was possible to further investigate the role of TLR4 in the adjuvant activity of PLY, in particular the PLYdependent production of IL-1@. Through these studies a surprising role for TLR4 in in vitro PLY-dependent cytokine production was discovered. Additionally, it was found that complement has an essential role in the PLY-dependent production of IL-1@. The role(s) of complement and IL-1@ in the adjuvant activity were further investigated using an in vivo immunization model and the biological basis for the difference in adjuvant activity of PLY and 76PLY was defined.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:616460 |
| Date | January 2014 |
| Creators | Dalziel, Catherine Ellen |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/5405/ |
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