Fig. 5: Reorientation of H2 HA enabled immunofocusing on HA-stem.
a, Insertion of oligoD into the head of H2 HA (A/Japan/305/1957) after residue S156 (Protein Data Bank ID: 2WRE). Three oligoD motifs on HA-head allowed for its tri-valent anchoring on alum. b, BLI binding analysis of H2 HA or reoH2HA with HA-stem-specific mAbs. c,d, Binding of stem-directed (MEDI8852 and FI6v3) or head-directed (8F8 and 8M2) mAbs to reoH2HA on streptavidin-coated (c) or alum-coated (d) ELISA plates. Data are presented as mean ± s.d. (n = 4 technical replicates). e, A three-dose immunization study with H2 HA or reoH2HA adjuvanted with alum/CpG in BALB/c mice. f,g, Serum H2 HA-specific (f) or stem-specific (g) IgG titers over time (n = 10 mice per group). Stem-specific IgG titers were measured with the H1-SS protein. Endpoint (week 12) antibody titers from the two groups are plotted on the right for comparison (each circle represents a single mouse). h, Cross-reactive binding of group 1 (H1 NC/99, H1 CA/09 and H5 VT/04) and group 2 (H3 VC/75, H7 NT/27 and H7 SH/13) HAs by week 12 antisera. Each circle represents a single mouse (n = 10 mice per group). i, Serum binding titers to group 1 and group 2 HAs in the presence of competing mAb—MEDI8852. Each circle represents a single mouse (n = 10 mice per group). Arrows indicate prime and boost immunizations in f,g. Dashed lines indicate the limit of quantification in f–i. Data are presented as geometric mean ± s.d. of the log10‐transformed values (f–i). Comparison of two groups was performed using the two-tailed Mann–Whitney U-test. P values of 0.05 or less were considered significant.
