Design of an RSV-based nanoparticle displaying a site II epitope–focused immunogen.
(A) Structural model of the prefusion RSVF trimer (PDBID: 4JHW), with two subunits shown as a gray surface and one subunit shown as a light blue cartoon representation with the epitope targeted by palivizumab (antigenic site II) highlighted in red. FFL_001 was previously designed to present the site II epitope in a computationally designed scaffold. FFLM was designed by evolution-guided resurfacing, where changes in amino acid identity are highlighted in blue. FFLM was genetically fused to the N terminus of RSVN, resulting in a high-density array of the epitope-scaffold, as shown by the structural model (based on PDBID: 2WJ8). (B) Kinetic binding affinities of site II–specific human nAbs measured by SPR. KDs were measured with RSVF/FFLM immobilized as ligand and antibody Fabs as analyte. Sensorgrams and fits are shown in S3 Fig. (C) DLS profiles for FFL_001 and FFLM fused to RSVN. The FFL_001-RSVN fusion protein formed higher-order oligomers in solution (66.6 nm of median diameter), whereas the resurfaced FFLM-RSVN fusion protein (NRM) was monodisperse, with a median diameter of 21 nm. (D) Analysis of the NRM nanoparticles by negative stain electron microscopy. Shown are the 2D class averages of two representative classes. Data are available in S1 Data. DLS, dynamic light scattering; Fab, antibody variable fragment; nAb, neutralizing antibody; PDB, Protein Data Bank; RSV, respiratory syncytial virus; RSVF, RSV fusion protein; RSVN, RSV nucleoprotein; SPR, surface plasmon resonance.