Generation and testing of a novel multimeric C3d based adjuvant

• Main Author: He, Yong-Gang
• Published: University of Newcastle Upon Tyne 2012
• Subjects: 541.2242
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.576537

C3d, a proteolytic fragment of complement component 3, is a ligand for complement receptor 2 (CR2) expressed by B cells. When antigen (Ag) coated with C3d cross-links CR2 and Ag specific B cell receptor, the threshold for B cell activation is lowered resulting in an enhanced response to Ag. This made C3d a prime adjuvant candidate but after many years of research this has failed to materialise. This project examines the ability of multimeric C3d to enhance the immune response to a target antigen utilising C4b-binding protein (C4BP) to provide a multimeric arrayed scaffold of human (h) C3d fused to Ag. Analysis of an extended hC3d-Hen Egg Lysozyme (HEL)-hC4BP array revealed HEL mis-folding compromised its functionality and secretion. Therefore, a new multimeric hC3d-Tetanus Toxin C Fragment (TICF)-hC4BP and appropriate multimeric controls were developed. These were successfully expressed and purified from mammalian cell cultures. Immunoassays confirmed recombinant proteins self-formed into large multimeric structures and individual domains were in the correct conformation. Initial studies demonstrated that multimeric hC3d-TTCF array could bind to both h & mouse (m)CR2. Wild type mice were immunised with proteins but antibody response to the multimeric hC3d- TTCF were poor. Thorough analysis of construct binding to a range of mouse and human B cells revealed that whilst hC3d could specifically interact with hCR2 it did not bind mCR2. Immunisation of hCR2+ mice with the multimeric hC3d- TTCF was found to be equally poor and studies with hCR2+.C3-1- mice suggested that mouse C3 could compete with hC3d binding to hCR2 in vivo. These results have established no discernible interactions of mCR2 with recombinant hC3d and have added to existing concerns regarding the limitations of using multimeric C3d as an adjuvant. Indeed, the results indicate that multimeric arrayed C3d-Ag is inhibitory with respect to immune response that may provide future therapeutic applications.