The molecular basis of adjuvant activity of pneumolysin

Dalziel, Catherine Ellen (2014) The molecular basis of adjuvant activity of pneumolysin. PhD thesis, University of Glasgow.

Abstract

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.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Pneumolysin, adjuvant, macrophage, TLR4, IL-1 beta, complement.
• Subjects: Q Science > QR Microbiology; Q Science > QR Microbiology > QR180 Immunology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Supervisor's Name: Mitchell, Prof. Timothy J.
• Date of Award: 2014
• Depositing User: Ms Mary Anne Meyering
• Unique ID: glathesis:2014-5405
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 Jul 2014 13:27
• Last Modified: 25 Jul 2017 15:04
• URI: https://theses.gla.ac.uk/id/eprint/5405