Abstract
The use of C3d, the final degradation product of complement protein C3, as a “natural” adjuvant has been widely examined since the initial documentation of its immunogenicity-enhancing properties as a consequence of binding to complement receptor 2. Subsequently it was demonstrated that these effects are most evident when oligomeric, rather than when monomeric forms of C3d, are linked to various test protein antigens. In this study, we examined the feasibility of enhancing the adjuvant properties of human C3d further by utilizing C4b-binding protein (C4BP) to provide an oligomeric arrayed scaffold fused to the model antigen, tetanus toxin C fragment (TTCF).High molecular weight, C3d-containing oligomeric vaccines were successfully expressed, purified from mammalian cells and used to immunize groups of mice. Surprisingly, anti-TTCF antibody responses measured in these mice were poor. Subsequently we established by in vitro and in vivo analysis that, in the presence of mouse C3, human C3d does not interact with either mouse or even human complement receptor 2.These data confirm the requirement to develop murine versions of C3d based adjuvant compounds to test in mice or that mice would need to be developed that express both human C3 and human CR2 to allow the testing of human C3d based adjuvants in mouse in any capacity.
Highlights
Whilst it has been established for 40 years that complement (C) plays an important role in both initiating and shaping adaptive immunity (Pepys, 1972, 1974), it was not until the identification and characterization of B cell complement receptor 1 (CR1; CD35) and 2 (CR2; CD21) expression [reviewed in (Cooper et al, 1988; Hourcade et al, 1989)], that the molecular basis for this was fully understood (Fearon and Carter, 1995; Hebell et al, 1991)
Using CD19 to sort B cells from hPBMC, we found that specific binding of the various biotinylated hC4BP constructs to B cell CR2 was dependent on the presence of hC3d (Fig. 2b)
To examine a possible reason for this discrepancy, and in light of the increased binding of mC3d to hCR2 in surface plasmon resonance (SPR) analysis, we examined the binding of hC3d-TTCFC4BP to B cells isolated from hCR2 transgenic mice that had been crossed with mice lacking C3 (C3−/−)
Summary
Whilst it has been established for 40 years that complement (C) plays an important role in both initiating and shaping adaptive immunity (Pepys, 1972, 1974), it was not until the identification and characterization of B cell complement receptor 1 (CR1; CD35) and 2 (CR2; CD21) expression [reviewed in (Cooper et al, 1988; Hourcade et al, 1989)], that the molecular basis for this was fully understood (Fearon and Carter, 1995; Hebell et al, 1991). The generation of Cr2 gene knockout mice (along with other key C component knockout animals) confirmed the essential roles that C and CRs play in adaptive immune responses (Ahearn et al, 1996; Molina et al, 1996). It has been clear for some time that many components of the innate immune system act to activate and enhance the adaptive immune response, and can be considered to be “natural” adjuvants (Morgan et al, 2005). This was first documented when multiple copies of mouse (m)C3d, in a linear trimer, were attached to a soluble antigen, and shown to enhance antigenspecific responses in mice up to 10,000 fold (Dempsey et al, 1996); potentially being more potent than complete Freund’s adjuvant
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