Fig. 1: In vitro characterisation of HA clamp antigens.

A A schematic of the HA gene showing the design of HA, HA ectodomain with no trimerisation domain (HA Sol), HA with the foldon trimerisation domain (HA foldon) and HA with the molecular clamp trimerisation domain (HA clamp). FP fusion peptide, TM transmembrane domain, CT cytoplasmic domain. HA1 is shown in red and HA2 is shown in blue. B An illustration of the HA protein with the molecular clamp replacing the transmembrane domain. The structure of the six-helix bundle molecular clamp (PDB ID 3P30) is also illustrated. C Coomassie blue stained SDS-PAGE analysis of purified HAs from seasonal and zoonotic influenza A viruses. Molecular weight standards are indicated in kDa. D Oligomeric state analysis of HA proteins via size-exclusion chromatography on a Superdex 200 Increase 10/300 GL column. The molecular weight of the standards in kDa is shown. Grey boxes highlight trimeric HA and yellow boxes highlight monomeric HA. E Representative micrograph from negative-stain TEM of H1 clamp. F 2D class averages of H1 clamp and a 2D reprojection of a representative H1 protein (PDB ID 3LZG). G Representative micrograph from negative-stain TEM of H3 clamp and H 2D class averages of H3 clamp.