Lumazine Synthase Nanoparticles as a Versatile Platform for Multivalent Antigen Presentation and Cross-Protective...

Lumazine synthase (LS), a bacterial protein that self-assembles into 60-mer icosahedral virus-like nanoparticles, has emerged as a promising platform for nanoparticle-based drug delivery and vaccine design. However, detailed biophysical characterization of the LS nanoparticle vaccine has not been well-studied. In this study, we generated LS nanoparticles fused with domain B of protein A (pA-LS), enabling their binding to the hFc-tagged S1 domain of the SARS-CoV-2 spike protein harboring two critical mutations (E484K and D614G) associated with increased infectivity and antibody escape. Biophysical analysis, such as transmission electron microscopy (TEM), revealed an extended size (∼45 nm) compared with the empty particle (∼15 nm). Similarly, atomic force microscopy (AFM) and dynamic light scattering (DLS) analyses confirmed increases in height and diameter. The spike-decorated nanoparticles demonstrated multivalent surface presentation by binding to the ACE2 receptor with a speckle-like appearance. Immunization of mice with pA-LS-S1-hFc elicited neutralizing antibodies against SARS-CoV-2 and its variants. Further, immunization followed by a live SARS-CoV-2 challenge (Wuhan-Hu-1, B.1.617.2 (Delta), or B.1.1.529 (Omicron)) in K18-hACE2 transgenic mice significantly reduced the lung viral load and pathology. Additionally, we generated mosaic nanoparticles displaying spike proteins from two epidemic coronaviruses, SARS-CoV-1 and MERS-CoV, which exhibited binding to their respective cellular receptors, ACE2 and DPP4, with similar binding patterns. Immunization with these mosaic nanoparticles elicited cross-reactive neutralizing antibodies against SARS-CoV-1 and MERS-CoV pseudoviruses. Our proof-of-concept data demonstrate the versatility of the LS nanoparticle platform for antigen presentation, supporting the development of multivalent vaccine designs targeting diverse antigens and contributing to immunogen design strategies.