Engineering the ADDomer Nanoparticle Vaccine Scaffold for Improved Assembly and Enhanced Stability

Virus-like particles (VLPs) are promising platforms for next-generation vaccines due to their ability to present antigens in highly ordered, repetitive geometries emulating pathogen-associated patterns to elicit potent immune responses. The ADDomer is a synthetic dodecahedral VLP scaffold derived from the penton base protein (PBP) of human adenovirus serotype 3 (Ad3). PBP tolerates insertion of multiple antigenic epitopes in flexible surface-exposed loops, and spontaneously self-assembles <i>in vitro</i> into ADDomer nanoparticles, but faces limitations including incomplete assembly and susceptibility to preexisting antihuman adenovirus immunity. Here, we report two complementary engineering strategies to enhance ADDomer robustness. First, we developed a Chimpanzee adenovirus Y25-based ADDomer (CHIMPSELS) to circumvent preexisting antihuman adenovirus immunity, and introduced a point mutation to restore a motif critical for dodecahedron integrity. Second, we introduced targeted intersubunit disulfide bonds to reinforce particle assembly. High-resolution electron cryo-microscopy confirmed the formation of intact dodecahedral particles, revealing that disulfide bonds stabilize distinct conformations of the PBP N-termini. Differential scanning fluorimetry and dynamic light scattering demonstrated thermal stability and elevated aggregation onset temperatures in the disulfide-stabilized ADDomers, providing a scalable assay for screening ADDomer-based VLP constructs for vaccine development. Incorporation of validated immunogenic epitopes, including a SARS-CoV-2 receptor-binding motif segment and the Chikungunya E2EP3 peptide, demonstrated structural integrity and epitope display by the modified scaffolds. Our results establish a versatile, thermostable VLP platform with reduced susceptibility to preexisting immunity, improved particle integrity, and capacity for modular epitope presentation. This work advances the ADDomer toward practical applications in vaccine development and highlights engineering strategies that can be broadly applied to enhance the performance of protein-based VLP vaccines.