Year

2007

Degree Name

Doctor of Philosophy

Department

School of Biological Sciences - Faculty of Science

Abstract

Group A streptococcus (GAS) causes invasive and non-invasive diseases, as well as severe immune sequelae such as rheumatic fever and acute post streptococcal glomerulonephritis. Several surface proteins have been proposed as candidate vaccine antigens; this study examined the serum opacity factor (SOF) of GAS, a cell-surface protein which causes opalescence of human serum and mediates bacterial binding to fibronectin.

A molecular characterisation of SOF from common Australian and international isolates revealed that SOF exhibits considerable variation in the N-terminal domain of the protein. This extensive variation has not led to a distinct geographic segregation, suggesting that SOF may have evolved a number of SOF sequence types that are common throughout global populations. In this study, hexahistidyl-tagged fusion proteins encompassing full-length SOF, and domains of SOF encompassing opacity factor activity (rSOFΔFn) and fibronectin binding domains (FnBD), were used in the characterisation of the Aboriginal immune response to SOF. Anti-SOF serum IgG responses were found to be significantly higher (p < 0.001) in Aboriginal adults and children when compared to the non-Aboriginal adult group. The Aboriginal immune response against the FnBD of SOF was significantly reduced when compared to the response against the whole SOF protein and the SOFΔFn domain (p < 0.001). This pattern of immune response was also observed in rabbits immunised with recombinant SOF. However, even though the immune response against SOF is directed toward the N-terminal variable domain, this study has shown that parenteral immunisation with the rSOFΔFn domain stimulates an immune response that can protect against a lethal systemic challenge with a heterologous GAS strain, suggesting that the SOF protein from different GAS strains may contain common protective epitopes.

While SOF protects against systemic heterologous GAS challenge when administrated parenterally, this study found that mucosal immunisation with the SOF protein fails to protect against mucosal heterologous GAS challenge. As the most effective protection against subsequent challenge is achieved when SOF is administered parenterally, the capacity of SOF to bind and precipitate high density lipoprotein (a function known as opacity factor activity or OF activity) may preclude its use as a vaccine antigen in humans. This study generated mutant forms of recombinant SOF with reduced (100 fold) or abrogated OF activity, for use as vaccine antigens. However, the structural alterations introduced into the N-terminal SOF domain (SOFΔFn) by mutagenesis to abrogate OF activity, abolish the capacity of SOF to protect against lethal systemic GAS challenge in a murine model.

Mutant forms of purified SOFΔFn were also used to assess the contribution of OF activity to the pathogenic processes of cell adhesion and cell invasion. Latex beads coated with full-length rSOF75, rSOF75ΔFn or rFnBD, were used to show that adhesion to HEp-2 cells is mediated by both the SOFΔFn and FnBD domains of SOF. The HEp-2 cell binding displayed by the N-terminal SOFΔFn peptide is independent of OF activity. While the N-terminus of SOF does not directly mediate ntracellular uptake by epithelial cells, this domain enhances epithelial cell uptake mediated by full-length SOF, in comparison to the FnBD alone.

This study characterised SOF as a vaccine antigen of GAS, and showed for the first time that vaccination with SOF can protect against infection by a heterologous GAS serotype in an animal model. However, a number of deficiencies were identified that must be resolved before SOF can be applied as a vaccine antigen, including the OF activity of SOF and also the observation that SOF protects against GAS challenge only when administered parenterally.

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