Abstract:
There has been a shift in vaccine development from being based on killed or attenuated whole microorganisms to smaller, more specific subunit vaccines, including peptides. However, peptides by themselves are often poorly immunogenic, necessitating the need for an adjuvant or a specialised delivery system. A novel peptide delivery strategy, Pilvax, was developed in this thesis by expressing peptides within the serotype M1 group A streptococcus (GAS) pilus structure on the surface of Lactococcus lactis. In GAS, each fully assembled pilus structure consists of approximately 50-100 Spy0128 pilin subunits that are covalently linked by isopeptide bond formation. Genetically engineering a peptide into the Spy0128 subunit will result in the expression of hundreds of repeated copies of the peptide on a single pilus and thousands of copies across the surface of the bacterium. The complete GAS pilus structure can be expressed and assembled on the surface of L. lactis, a non-pathogenic bacterium which has generally recognised as safe (GRAS) status. L. lactis is a promising antigen delivery vehicle, having been used to deliver numerous vaccine candidate antigens to both mucosal and systemic sites. Six loop regions and the N-terminus of the Spy0128 pilin were selected as possible sites where a peptide could be engineered into. However, peptides could only be engineered into two of the loop regions without affecting the polymerisation of the pilus on the surface of L. lactis. To show proof of principle, a peptide consisting of ovalbumin from positions 323 to 339 (OVA323-339) was engineered between the βE and βF loop of Spy0128 and the peptide-linked pilus was expressed on the surface of L. lactis. Incorporation of OVA323-339 into the pilus structure was demonstrated by Western blot analyses of cell wall proteins and by flow cytometry. Intranasal immunisation of mice with L. lactis expressing pilus-linked OVA323-339 results in production of both a systemic and mucosal antibody response against ovalbumin. The feasibility of using Pilvax to deliver a structural epitope was then investigated. The J14 peptide, a chimeric peptide derived from the carboxy-terminal C-repeat region of the GAS M protein, was chosen to be engineered into Pilvax. The peptide contains a conformationally restricted B cell epitope fused to coil-promoting moieties from the yeast GCN4 protein to maintain the original coiled-coil structure. The J14 peptide engineered between the βE and βF loop of Spy0128 showed incorporation of J14 into pili, expressed on the surface of L. lactis, as demonstrated by Western blot analyses of cell wall proteins. However, intranasal immunisation of mice with L. lactis expressing pilus-linked J14 only induced a weak J14-specific antibody response and the antibodies elicited could not recognise and bind the native M protein. This suggests that the alpha-helical conformation of J14, required for the peptide to be an effective vaccine candidate, is disrupted when it is engineered into a loop region of Spy0128. Thus, Pilvax may provide an alternative strategy to allow safe and effective delivery of vaccine peptides to mucosal sites, but may be limited to conformation-independent peptides.