All antigens and antibodies were expressed in Expi-293F cells. Expi-293F cells were cultured at 37 °C under constant shaking (120 r.p.m.) in a humidified CO2 (8%) incubator. Expi-293F cells were transfected at a density of 3–4 × 10^6 cells/mL. For 200-ml transfection of antigen proteins, the transfection mixture was made by adding 120 µg of plasmid DNA to 20 ml of expression media, followed by dropwise addition of 260 µl of FectoPRO with vigorous mixing. For antibody production in 200 ml of Expi-293F cells, the transfection mixture contained 60 µg of LC plasmid DNA and 60 µg of HC plasmid DNA. Transfection mixtures were incubated at room temperature for 10 min before being transferred to Expi-293F cells. D-glucose (4 g.L−1) and valproic acid (3 mM) were added to the cells immediately after transfection to increase recombinant protein production. Cells were boosted again with D-glucose 3 d after transfection and harvested on day 4 by centrifugation at 7,000g for 5 min. The supernatant was filtered through a 0.22-µm membrane for subsequent purification.

A major challenge in creating universal influenza vaccines is to focus immune responses away from the immunodominant, variable head region of hemagglutinin (HA-head) and toward the evolutionarily conserved stem region (HA-stem). Here we introduce an approach to control antigen orientation via site-specific insertion of aspartate residues that facilitates antigen binding to alum. We demonstrate the generalizability of this approach with antigens from Ebola, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses and observe enhanced neutralizing antibody responses in all cases. We then reorient an H2 HA in an ‘upside-down’ configuration to increase the exposure and immunogenicity of HA-stem. The reoriented H2 HA (reoH2HA) on alum induced stem-directed antibodies that cross-react with both group 1 and group 2 influenza A subtypes. Electron microscopy polyclonal epitope mapping (EMPEM) revealed that reoH2HA (group 1) elicits cross-reactive antibodies targeting group 2 HA-stems. Our results highlight antigen reorientation as a generalizable approach for designing epitope-focused vaccines.