Tailored Design of Protein Nanoparticle Scaffolds for Multivalent Presentation of Viral Glycoprotein Antigens
George UedaiD, Aleksandar AntanasijeviciD, Jorge A. FallasiD, William SheffleriD, Jeffrey CoppsiD, Daniel EllisiD, Geoffrey B. HutchinsoniD, Adam P. MoyeriD, Anila YasmeeniD, Yaroslav TsybovskyiD, Young‐Jun ParkiD, Matthew J. BickiD, Banumathi SankaraniD, Rebecca A. GillespieiD, Philip J. M. BrouweriD, Peter H. Zwart, David VeesleriD, Masaru KanekiyoiD, Barney S. GrahamiD, Rogier W. SandersiD, John P. MooreiD, Per Johan KlasseiD, Andrew B. WardiD, Neil P. KingiD, David A. BakeriD
Abstract
The adaptive immune system is highly sensitive to arrayed antigens, and multivalent display of viral glycoproteins on symmetric scaffolds has been found to substantially increase the elicitation of antigen-specific antibodies. Motivated by the considerable promise of this strategy for next-generation anti-viral vaccines, we set out to design new self-assembling protein nanoparticles with geometries specifically tailored to scaffold ectodomains of different viral glycoproteins. We first designed and characterized homo-trimers from designed repeat proteins with N-terminal helices positioned to match the C termini of several viral glycoprotein trimers. Oligomers found to experimentally adopt the designed configuration were then used to generate nanoparticles with tetrahedral, octahedral, or icosahedral symmetry. Examples of all three target symmetries were experimentally validated by cryo-electron microscopy and several were assessed for their ability to display viral glycoproteins via genetic fusion. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles display conformationally intact native-like HIV-1 Env, influenza hemagglutinin, and prefusion RSV F trimers in the predicted geometries. This work demonstrates that novel nanoparticle immunogens can be designed from the bottom up with atomic-level accuracy and provides a general strategy for precisely controlling epitope presentation and accessibility.
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In-depth summary
Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (30 Jan 2020) · CC BY 4.0
Designed trimers matched to viral glycoprotein termini yielded tetrahedral, octahedral and icosahedral nanoparticles with cryo-EM-verified structures that display native-like HIV Env, influenza HA and RSV F.
Earlier computationally designed nanoparticle immunogens were built from subunits derived from natural oligomers, leaving structural control incomplete. Scaffolds designed from scratch with N termini matched to antigen C termini could display antigens with less distortion near their base.
Key results
ShownComputational design of C3 homotrimers produced many soluble proteins, and a subset adopted the intended oligomeric state. Crystal structures of two trimers matched their design models closely.
23 designs tested; 21 expressed soluble; 11 formed the intended oligomer by SEC-MALS. Crystal structures of 1na0C3_2 and 3ltjC3_1v2 at 2.6 and 2.3 Å, heavy-atom RMSD to design 1.4 and 0.8 Å. · Structural characterization of designed trimers
ShownPairing validated trimers with partner oligomers gave a low but real yield of correctly assembling two-component nanoparticles, spanning tetrahedral, octahedral and icosahedral symmetry.
53 designs tested; 24 co-eluted on SDS-PAGE; 19 failed to form assemblies of expected size or were heterogeneous; 5 (T33_dn2, T33_dn5, T33_dn10, O43_dn18, I53_dn5) were monodisperse by SEC-MALS with SAXS matching design models. NS-EM maps matched the models. · Generation of two-component nanoparticles from…; Structural Characterization of Designed…
ShownCryo-EM of one nanoparticle per target symmetry showed excellent agreement between the refined structures and the Rosetta design models.
Resolutions 3.9, 4.5 and 5.3 Å for T33_dn10, O43_dn18 and I53_dn5; asymmetric-unit backbone RMSD to design 0.65, 0.98 and 1.3 Å, respectively. · Structural Characterization of Designed…; Cryo-EM Data Processing
ShownViral glycoproteins fused to the trimeric components were displayed on assembled particles without disturbing the scaffold core. RSV F and HA antigenicity was consistent with native folding. Linker flexibility blurred the antigen density.
BG505 SOSIP on T33_dn2, T33_dn10 and I53_dn5 showed unchanged cores by NS-EM. DS-Cav1–I53_dn5 bound D25, Motavizumab and AM14 (ELISA) like soluble DS-Cav1 with foldon. HA–I53_dn5 gave wild-type-like head and stem mAb binding (BLI). · Characterization of Viral…
SuggestedScaffold geometry changed epitope accessibility on BG505 SOSIP. Base-proximate epitopes were more accessible on tetrahedral T33_dn2 than on the published icosahedral I53-50.
SPR nanoparticle/trimer mole ratio ~1 for PGT145, PGT122, 2G12 and VRC01; reduced for ACS202, VRC34, PGT151, 3BC315 and 11B; binding of 12N obliterated. VRC34 and PGT151 showed nearly 3-fold higher accessibility on T33_dn2 than I53-50 (historic data; 2-4 replicates). · Control over BG505 SOSIP Epitope Accessibility…; Surface Plasmon Resonance (SPR) Analysis of…; Figure legends
Methods, data and reproducibility
Hierarchical Rosetta design: C3 docking of repeat proteins scored by RPX and screened for N-terminus spacing matching antigen C termini, then TCdock pairing with partner oligomers and interface design. Validation used SEC-MALS, SAXS, crystallography, NS-EM, cryo-EM and SPR.
- Designed C3 homotrimers from repeat protein scaffolds, expressed in E. coli; two crystal structures · E. coli (expression host) · n = 23 designs selected · PDB 6V8E, 6VEH
- Two-component tetrahedral, octahedral and icosahedral nanoparticle designs; NS-EM maps of nanoparticles and antigen fusions · n = 53 designs (11 tetrahedra, 21 octahedra, 21 icosahedra) · EMDB 21162-21171
- Cryo-EM structures of T33_dn10, O43_dn18 and I53_dn5 nanoparticles · n = 3 nanoparticles · PDB 6VFH, 6FVI, 6VFJ; EMDB 21172, 21173, 21174
- Target antigen structures used for termini matching: HIV-1 BG505 SOSIP, influenza H1 HA, RSV F DS-Cav1 · HIV-1, influenza virus, RSV (antigens expressed in HEK293F) · PDB 5VJ6, 5KAQ, 5TPN
- SPR of BG505 SOSIP–T33_dn2A trimer and T33_dn2 nanoparticle against a panel of anti-Env mAbs, compared with historic I53-50 data · HIV-1 · n = 2-4 replicates
- Methods & tools
- Rosetta, RPX scoring, sic_axle, TCdock, SEC-MALS, SAXS, Relion 3.0, cryoSPARC 2.9.0, MotionCor2, GCTF v1.06, BIAcore 3000 SPR, Coot
- Reagents & environment
- cryoSPARC 2.9.0, Relion 3.0 for cryo-EM (Relion 2.1 for NS-EM 3D steps), GCTF v1.06, Relion 1.4 and SPIDER for DS-Cav1–I53_dn5 2D classification.
- Reproducibility gaps
- No code repository URL is given; design sequences are in supplementary tables. Particle-picking software used for DS-Cav1–I53_dn5 is in-house and unpublished. Accessions are PDB and EMDB IDs only.
Limitations (4)
- Free nanoparticle components were seen in the T33_dn5 sample, suggesting a tendency to disassemble. · stated by the authors
- Antigen trimers were less well resolved than nanoparticle cores, attributed to short flexible linkers; the GG linker in DS-Cav1–I53_dn5 gave poor glycoprotein definition in 2D classes. · stated by the authors
- No immunization or B cell data are presented, so the claimed value for eliciting epitope-targeted responses is untested; only structure and antigenicity were measured. · noted in this summary
- The accessibility comparison with I53-50 relies on historic data from a prior study and one new scaffold (T33_dn2), with only 2-4 replicates; sample-to-sample differences may confound it. · noted in this summary
Who should read:Read this if you design protein nanoparticle scaffolds or vaccine immunogens and want experimental success rates and cryo-EM validation for geometry-tailored antigen display.
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