Single-component self-assembling protein nanoparticles displaying stabilized prefusion-closed hemagglutinin trimers for influenza vaccine development

ABSTRACT Current influenza vaccines primarily target hemagglutinin (HA), the major 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, pose major challenges to the development of broadly protective influenza vaccines. Here, we stabilize HA in the prefusion-closed conformation through rationally selected amino acid substitutions. One approach targets a conserved residue across multiple influenza A subtypes (H1, H3, H5, and H7) and both influenza B lineages, providing a unified and broadly applicable framework for HA stabilization to support vaccine development. Using H1 and H3 as test cases, stabilized HA trimers were displayed on 24- and 60-mer single-component self-assembling protein nanoparticles (SApNPs) to enhance lymph node trafficking and immunogenicity. Compared with soluble trimers, HA-presenting SApNPs exhibited prolonged retention in lymph node follicles and elicited robust germinal center (GC) responses, key hallmarks of effective virus-like particle (VLP) vaccines. In mice, these SApNPs induced strong humoral immunity and conferred protection against homologous viral challenge. Furthermore, glycan engineering to enrich oligomannose content augments vaccine-induced NAb responses. Together, these findings provide mechanistic insights and establish design principles for next-generation HA-based influenza vaccines targeting both seasonal and pandemic strains. ONE-SENTENCE SUMMARY Rational HA design and nanoparticle display guide next-generation strategies for influenza vaccine development