Development of thermostable vaccine conjugates based on a staphylococcal immune evasion protein

Despite the successes of global vaccination programs, infectious diseases continue to claim or devastate millions of lives each year. Reductions in this number will require dramatic improvements in vaccines via strategies including the development of efficacious adjuvants which augment vaccine-induced immune responses and elimination of the refrigerated cold chain through vaccine thermostabilisation. Recent investigations have revealed Staphylococcus aureus binder of immunoglobulin (Sbi) harbours potential as a novel adjuvant as (1) Sbi III-IV has the unique ability to encourage the consumptive cleavage of the central complement component C3 to C3b, iC3b and the natural molecular adjuvant C3d, and (2) C3d-opsonised antigens promote C3d-complement receptor 2 (CR2) and antigen-B cell receptor (BCR) coligation on B cells and generate enhanced immune responses. In addition, ensilication, a method that encases proteins in a resistant silica cage, has been shown to physically prevent the thermal denaturation of a number of model proteins and has potential for vaccine thermostabilisation. The research presented in this thesis reveals Sbi III-IV-mediated C3 consumption involves the formation of dimeric tripartite complexes with C3b and Factor H-related proteins and induces opsonisation of tuberculosis antigen Ag85b and anthrax protective antigen with C3 activation products. Opsonised Ag85b is shown to elicit a rapid and heightened humoural immune response in a C3 and CR2 dependent manner in vivo, establishing the application of Sbi III-IV in enhancing the immunogenicity of antigens key to the development of novel vaccine candidates. Subsequent analyses uncover the thermal lability of the Sbi III-IV adjuvant, antigen Ag85b and a newly-developed Sbi III-IV-Ag85b conjugate and provide evidence of the utility of ensilication in improving the thermal stability of these vaccine-relevant constructs, thus demonstrating its use in a vaccine setting for the first time. Finally, C3d is shown to form disulphide-linked dimers that crosslink CR2, a new discovery with further potential implications for vaccine design. In the future, Sbi III-IV and dimeric C3d could serve as robust adjuvants that potentiate immune responses induced by multiple vaccines and ensilicationmediated thermostabilisation could eliminate the need for the cold chain and thereby improve the global distribution of safe and effective vaccines.