Structure-based design and immunogenicity of an H5 hemagglutinin stem nanoparticle vaccine candidate.

Highly pathogenic avian influenza viruses (HPAIVs), particularly the H5N6 subtype, pose a serious global public health threat. Conventional influenza vaccines primarily target the highly variable globular head of hemagglutinin (HA), resulting in limited breadth of protection. Here, we employed a structure-guided vaccine design strategy to develop a self-assembling nanoparticle immunogen targeting conserved epitopes within the HA stem region. The HAstem antigen was rationally engineered to incorporate major neutralizing epitopes and further stabilized through the introduction of a GCN4 trimerization motif, engineered disulfide bonds, and prefusion conformation optimization. Using the I53_dn5 platform, HAstem was displayed in a highly ordered and multivalent array on the surface of icosahedral nanoparticles. Cryo-electron microscopy at 4.99 Å resolution confirmed the structural integrity of the nanoparticle core and the successful assembly of the vaccine construct. Immunization with HAstem-I53_dn5 elicited robust HAstem-specific antibody responses, with endpoint titers reaching 1:12,800, significantly exceeding those induced by both trimeric antigen and inactivated whole-virus vaccines. In addition, the vaccine induced diverse HA stem-specific IgG subclasses, potent FcγRIV-binding antibodies, and cross-reactive antibodies preferentially targeting H5 subtype HA antigens. In summary, these findings establish the structural and immunogenic characteristics of the HAstem-I53_dn5 nanoparticle and support its further development as an H5-focused HA stem nanoparticle vaccine candidate.