Utilisation of staphylococcal immune evasion protein Sbi as a novel vaccine adjuvant

Abstract

42Co--ligation of the B cell antigen receptor with complement receptor 2 on B--cells 43 via a C3d--opsonised antigen complex significantly lowers the threshold required 44 for B cell activation. Consequently, fusions of antigens with C3d polymers have 45 shown great potential in vaccine design. However, these linear arrays of C3d 46 multimers do not mimic the natural opsonisation of antigens with C3d. Here we 47 investigate the potential of using the unique complement activating 48 characteristics of Staphylococcal immune--evasion protein Sbi to develop a 49 pro--vaccine approach that spontaneously coats antigens with C3 degradation 50 products in a natural way. We show that Sbi rapidly triggers the alternative 51 complement pathway through recruitment of complement regulators, forming a 52 tripartite complex that acts as a competitive antagonist of factor H, resulting in 53 enhanced complement consumption. These functional results are corroborated 54 by the structure of this complement activating Sbi--III--IV:C3d:FHR--1 complex. 55Finally, we demonstrate that Sbi, fused with Mycobacterium tuberculosis antigen 56 Ag85b, causes efficient opsonisation with C3 fragments, thereby enhancing the 57 immune response significantly beyond that of Ag85b alone, providing proof of 58 concept for our pro--vaccine approach. 59 antigens by C3d at a molecular level and do not always enhance immune 82 responses [13]. After activation of C3, C3b attaches directly to the antigen 83 surface via the reactive thioester on the convex face of the protein's thioester 84 domain (TED). In the presence of complement regulators (factor I (FI) and its 85 co--factors, such as factor H (FH) and CR1) this is rapidly converted to iC3b and 86 then to C3d, exposing the concave CR2 binding site of the TED fragment away 87 from the antigen surface [14]. It is likely that multiple iC3b/C3d molecules attach 88 to complex antigens/pathogen surfaces during the initial activation phases of 89 complement, creating high--avidity binding site for complement fragment 90 receptors. 91In the last two decades, structural biology has helped to unveil many of the 92 molecular aspects that are crucial for the activation and regulation of the 93 complement system. Most notable are the crystal structures of the central 94 complement component activation states, native C3 [15], activated C3b and 95 inactive C3c [16]. The structure of C3b in complex with factors B and D [17] 96 subsequently revealed a detailed view of the alternative pathway C3 convertase 97 assembly and its activation, leading to the amplified cleavage of C3 molecules 98 that result in opsonisation and clearance of microbial pathogens and host debris. 99 The covalent attachment of C3b to surfaces does not discriminate between self 100 or non--self surfaces and requires tight regulation to protect host surfaces. 101 Structures of C3b in complex with FH domains 1--4 [18] and domains 19--20 [19, 102 20] provided insights into protection of host cells [21] and demonstrated how 103 5 factor H--related prote...