Generation and functional analysis of an octameric C3d construct to act as a molecular adjuvant in vaccine design

Abstract

This thesis investigated two separate theoretical advancements for vaccine design: (1) present antigen in a self-forming natural C4b binding protein-based octameric array (to engage multiple adjacent B-cell receptors or BCR) and (2) combined with fusion to a molecular adjuvant C3d or C3dg, to cross-link C3d-receptor (complement receptor 2 (CR2) CD21). Initially, yellow fluorescent protein test antigen was evaluated, but only to monitor intracellular and extracellular protein levels for a range of C3 forms: C3dg (smallest covalently bound CR1/factor I processed form of activated C3) or C3d (non-complement enzyme processed C3), as well as consequence of mutating the internal thioester bond (C988S, 'C3dsg7 C3ds) that may confound recombinant production of C3dg/C3d fragments. The test antigen was then switched to hen egg lysozyme (HEL), with the hope that spider-like HEL octamer arrays would be expressed with the C3dg/C3d fused proteins at the outermost reach. Mutating the thioester bond increased expression for both C3 forms, and C3dsg-fusion octamers expressed at the highest levels, although C3ds had the highest binding for human CR2. Receptor binding was considered more important for vaccine function, leading to the prototype vaccine, hC3ds-HEL-Oct. Further dissection of the CR2-C3d interaction utilised cell-based assays and surface plasmon resonance (SPR) analysis, using dimeric immunoglobulin Fc-fusion recombinant forms of human C3d, C3dg and murine C3d. All C3dg/C3d Fc proteins bound strongly, although cross-species murine C3d Fc binding to human CR2 was 2-3 times higher than human counterparts. In vivo production of total immunoglobulin G (IgG) and IgM levels was measured, following injection of hC3ds-HEL-Oct, either as DNA or purified protein, and showed successful initiation of a HEL-specific immune response. Data suggested that CpG sequences may have a detrimental impact on vaccine function. These results highlight the power and flexibility of the octamer approach as well as C3d and their applications in vivo and in vitro.