Nanostructured Display of the Conserved Neuraminidase HCA2 Epitope by Nanocomplexes and Virus-Like Particles for Influenza Vaccine Development

Influenza neuraminidase (NA) has emerged as a promising target for universal influenza vaccine development because it undergoes less antigenic drift than hemagglutinin. Among the diverse NA subtypes of influenza A and B viruses, a highly conserved sequence adjacent to the enzymatic active site, termed highly conserved area 2 (HCA2), has been identified as an exceptionally conserved antigenic region. Here, we developed and evaluated two nanostructured vaccine platformsself-assembling protein nanocomplexes (NCXs) and virus-like particles (VLPs)displaying conserved HCA2 epitopes from influenza A and B NAs to enhance HCA2 immunogenicity. Intranasal immunization with HCA2 NCXs elicited anti-HCA2 IgG antibodies, predominantly of the IgG1 subclass, but induced low or undetectable T-cell responses in the spleen. In contrast, intranasal immunization with HCA2 VLPs elicited not only anti-HCA2 IgG antibodies but also anti-N1, anti-N2, and anti-BN IgG antibodies in sera, as well as detectable anti-N1 and anti-BN IgA antibodies in bronchoalveolar lavage fluids. HCA2 VLPs also induced substantially stronger CD4+ helper and CD8+ cytotoxic T-cell responses, particularly including both their central and effector memory subsets. Notably, only HCA2 VLP immunization, but not HCA2 NCX immunization, conferred protection against a lethal H1N1 virus challenge. These findings demonstrate that the architecture of HCA2 antigen presentation influences the quality and magnitude of the immune response. HCA2 VLPs elicited robust humoral and cellular immune responses and were associated with protection against a lethal influenza virus challenge. These results highlight the importance of nanostructure platforms in the development of conserved-epitope vaccines and support HCA2-displaying VLPs as a promising platform for next-generation influenza vaccine development.