RBS is structurally conserved between strains

A) Structure of the H1SI06 hemagglutinin (PDB: 6OC3) epitope highlighting the four major RBS regions. B) Sequence conservation visualisation between H1SI06 and H5VN04, and H1SI06 and H3HK68. C) Structural conservation between H1SI06 and H5VN04, and H1SI06 and H3HK68, illustrated as RMSDCA distance. D) AF2 predicted model of H1H5_head and H1H3_Head. Replaced segments from H5VN04 and H3HK68 are highlighted in green and blue, respectively. E-F) Crystal structure of the H1H5_Head and H1H3_Head chimera in complex with FluA20 Fab, where the sequence transplanted segments are coloured green and blue, respectively. The right panel shows the chimera (grey) overlaid on the H1SI06 WT (wheat) and zoomed in on the main RBS segments showing structural similarity of 3 segments and variability of loop 130 compared to H5VN04 WT apo and H3HK68 WT in complex with FluA20 (PDB: 4BGW and 6OCB, respectively).

Full length RBS chimera bind subtype-specific antibodies

A-B) Structure of the H1SI06 hemagglutinin RBS epitope highlighting additional residues included in the full-length chimera. Transplanted residues by H5VN04 and H3HK68 are highlighted in green and blue, respectively. Left panel shows zoom in on RBS highlighting major contact residues added in H1H5_FL2 and H1H3_FL2 C-D) BLI affinity measurements for H5VN04, H1H5_FL, H1H5_FL2, and H1SI06 against an H5 RBS-subtype-specific antibody 13D4 IgG and H3HK68, H1H3_FL, H1H3_FL2, and H1SI06 against an H3 RBS-subtype-specific antibody F045-92 IgG, respectively.

Heterotrimeric hemagglutinin chimera display cross-subtype and cross-group antibody binding

A) Linear construct of H1_H1H5_H1H3 chimeric heterotrimer and schematic. Left panel shows AlphaFold3 prediction of trimeric hemagglutinin consisting of the H1SI06 (grey), H1H5_FL2 (green), and H1H3_FL2 (blue). B-D) BLI affinity measurements against H1, H5, and H3 RBS-specific antibodies CH65 IgG, 13D4 IgG and F045-92 IgG, respectively.

RBS chimera elicit cross-reactive antibodies

A) 6 groups of mice (n=5) were immunised three times in a homologous boost scheme with H1SI06, H5VN04, H3HK69, H1H5_FL2, H1H3_FL2, or H1_H1H5_H1H3 heterotrimer. B-D) Binding titers of the mouse serum IgG collected at day 56 post-prime against H1, H5 or H3 head domains determined by enzyme-linked immunosorbent assay (ELISA). The median values are shown as lines.

Cross-group neutralization elicited by RBS chimera

Neutralising activity of mouse immune sera raised against full length hemagglutinin WT, chimera, or heterotrimer against A) H1N1 (A/Brisbane/59/2007) pre-pandemic strain, B) H5N1 (NIBRG-14, carrying HA derived from A/Vietnam/1194/2004), C) H3N2 (A/X-31). The logarithm of IC50 values with standard error of the mean (s.e.m.) are shown. The threshold line represents day 0.