Research into a vaccine against the human pathogen group A streptococcus (GAS) has been ongoing for many years. The majority of this research has focussed on the M-protein, the major virulence determinant of GAS. However, vaccine development has been hindered by serotypic diversity of the M-protein, and the potential for epitopes of this protein to induce immune responses that cross react with host proteins. In more recent years these hurdles have been overcome with several parenterally delivered vaccine candidates progressing into clinical trials. In an effort to increase vaccine efficacy, next generation GAS vaccine design has been directed towards the induction of mucosal immunity. In this thesis the development of a new polytope vaccine, designed to be expressed on the surface of the oral commensal organism S. gordonii, as a method for mucosal vaccine delivery, is described. The two protective antigens chosen for inclusion into the polytope vaccine candidate were the conformationally restricted epitope from the M-protein (J14), and a fragment of the streptococcal fibronectin binding protein (SfbI) called H12. The combination of the two antigens in a single polytope construct was designed as a two pronged approach to vaccine efficacy. In support of the inclusion of J14 into the vaccine polytope, an in silico approach was undertaken to confirm its safety in populations with diverse genetic backgrounds. ProPred, RANKPEP and HLABIND algorithms failed to predict significant binding between the M-protein specific regions of J14 and class I and class II binding alleles. A single peptide within J14 was predicted to bind to HLA class I allele B_2705. These data suggests that J14 is unlikely to induce cross-reactive immune responses, and will be safe for use in humans. As a prelude to the development of the live mucosal delivery system, a series of recombinant fusion proteins, containing J14 and H12 in different orientations, were constructed and their immunogenicity compared. Surprisingly, when expressed as a fusion with H12, J14 proved to be a poor immunogen. Therefore, in an effort to increase the immunogenicity of J14, modifications were made to the recombinant fusion proteins to promote their coiled-coil structure. A recombinant protein containing two J14 moieties in tandem, with an amino acid sequence optimised for coiled-coil formation was constructed (JJo). Circular dichroism confirmed that JJo did indeed have an increased propensity for alpha-helical conformation. When JJo was expressed as a fusion with H12, an alanine spacer was incorporated at the junction of the two antigens to reduce the influence of H12 on the alpha-helical conformation of J14. In contrast to the original fusion proteins, JJoH12 (containing the optimised tandem J14 and the alanine spacer) was successful in stimulating a humoral response to both J14 and H12 in a murine model.
To express JJoH12 in S. gordonii, a genetic system previously designed for the integration of heterologous genes into the bacterial chromosome was utilised. This system uses a plasmid based integration strategy (pSMB55) which results in the surface anchorage of the heterologous protein as a fusion with an M-protein derived signal sequence and anchor domain. The gene encoding JJoH12 was cloned into pSMB55 and subsequently incorporated into the chromosome of S. gordonii GP251 through homologous recombination. A second recombinant strain, in which the M6 anchor domain was replaced by the anchor domain of SfbI, was also constructed. Immunofluorescence microscopy and flow cytometry confirmed expression of JJoH12 on the surface of both recombinant S. gordonii strains. Subcutaneous delivery of these strains confirmed that both antigens were immunogenic in a murine model. Following mucosal delivery, the recombinant strains colonised the upper respiratory tract of mice for at least five weeks. While no mucosal antibody response to JJoH12 was apparent, an antibody response to S. gordonii could be detected. The inability of the recombinant strains to induce J14 and H12 specific antibody responses may be overcome by optimisation of the mucosal vaccination regime.