Single-component self-assembling protein nanoparticles displaying stabilized prefusion-closed hemagglutinin trimers...

Current influenza vaccines primarily target hemagglutinin (HA), the major viral surface glycoprotein and principal determinant of neutralizing antibody (NAb) responses. However, antigenic drift and shift, together with HA's intrinsic metastability and low-pH sensitivity, limit broad and durable vaccine protection. Here, we stabilize HA in its prefusion-closed conformation through structure-guided amino acid substitutions. Targeting a conserved residue in the HA2 central triple helix-N95 in influenza A and Q95 in influenza B-provides a core design principle for modulating HA metastability across influenza A subtypes and both influenza B lineages, although the effects vary across viral groups. Using H1 CA09 and H3 HK68 as representative strains, we display stabilized HA trimers on 24-mer ferritin and 60-mer multilayered single-component self-assembling protein nanoparticles (SApNPs). In mice, HA-presenting SApNPs exhibit prolonged retention in lymph node follicles and elicit more robust germinal center responses compared with soluble trimers. Stabilized HA trimers and SApNPs induce functional antibody responses and confer varying levels of protection against homologous, heterologous, and cross-lineage viral challenges. Glycan modification enhances NAb induction or protection in some settings. Together, these findings provide mechanistic insights into HA metastability and establish a rational design framework for next-generation HA-based influenza vaccines.