Fig. 1

The spy-VLP antigen display platform. a Three types of spy expressing VLPs were constructed by genetic fusion of SpyTag or SpyCatcher to the virus-like particle (VLP)-forming AP205 capsid protein. (1) “SpyTag-VLP” had the SpyTag fused to the N-terminus of the AP205 capsid protein and present 180 potential SpyCatcher-antigen binding motifs (2) “2xSpyTag-VLP” had SpyTag fused to both the N- and C-terminus of the AP205 capsid protein and present 360 potential SpyCatcher-antigen binding motifs; (3) “SpyCatcher-VLP” had SpyCatcher fused to the N-terminus of the AP205 capsid protein and present 180 potential Spytag-antigen binding motifs. b Transmission electron microscopy (TEM) images showing the SpyTag-VLP, 2xSpyTag-VLP and SpyCatcher-VLP. Purified spy-VLP samples were placed on carbon, adsorbed to a grid and negatively stained with 2 % phosphotungstic acid. Scale bar 50 nm. Images show uniform, non-aggregated particles of approximately 30 nm (SpyTag-VLP and 2xSpyTag-VLP) and 42 nm (SpyCatcher-VLP). c Reduced SDS-PAGE gels loaded with VLP vaccines demonstrating that vaccine proteins had formed covalent bonds to the AP205 capsid protein. Left panel shows that mixing of SpyTag-VLPs with SpyCatcher-IL-5 resulted in three protein bands corresponding to the size of an antigen-VLP capsid protein conjugate (48 kDa) (top), uncoupled vaccine antigen (33 kDa) (middle) and unconjugated SpyTag-VLP capsid protein (16.5 kDa) (bottom). The middle panel shows that mixing of 2xSpyTag-VLP with SpyCatcher-IL-5 resulted in four protein bands representing; a conjugate of two vaccine antigens bound to each end of a 2xSpyTag-VLP capsid protein (83 kDa), a conjugate of the 2xSpyTag-VLP capsid protein and a single vaccine antigen (48 kDa), uncoupled vaccine antigen (33 kDa) and unconjugated 2xSpyTag-VLP capsid protein (18.5 kDa). The left panel shows that mixing of SpyCatcher-VLP with PD-L1-SpyTag resulted in three protein bands representing; an antigen-VLP capsid protein conjugate (50 kDa), uncoupled vaccine antigen (33 kDa) and unconjugated SpyCatcher-VLP capsid protein (27 kDa)