1. Computational and Systems Biology
  2. Immunology and Inflammation
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Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens

  1. George Ueda
  2. Aleksandar Antanasijevic
  3. Jorge A Fallas
  4. William Sheffler
  5. Jeffrey Copps
  6. Daniel Ellis
  7. Geoffrey B Hutchinson
  8. Adam Moyer
  9. Anila Yasmeen
  10. Yaroslav Tsybovsky
  11. Young-Jun Park
  12. Matthew J Bick
  13. Banumathi Sankaran
  14. Rebecca A Gillespie
  15. Philip JM Brouwer
  16. Peter H Zwart
  17. David Veesler
  18. Masaru Kanekiyo
  19. Barney S Graham
  20. Rogier W Sanders
  21. John P Moore
  22. Per Johan Klasse
  23. Andrew B Ward  Is a corresponding author
  24. Neil P King  Is a corresponding author
  25. David Baker  Is a corresponding author
  1. Department of Biochemistry, University of Washington, United States
  2. Institute for Protein Design, University of Washington, United States
  3. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, United States
  4. International AIDS Vaccine Initiative Neutralizing Antibody Center, the Collaboration for AIDS Vaccine Discovery (CAVD) and Scripps Consortium for HIV/AIDS Vaccine Development (CHAVD), The Scripps Research Institute, United States
  5. Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States
  6. Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, United States
  7. Electron Microscopy Laboratory, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, United States
  8. Berkeley Center for Structural Biology, Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley Laboratory, United States
  9. Amsterdam UMC, Department of Medical Microbiology, Amsterdam Infection & Immunity Institute, University of Amsterdam, Netherlands
  10. Center for Advanced Mathematics in Energy Research Applications, Computational Research Division, Lawrence Berkeley Laboratory, United States
  11. Howard Hughes Medical Institute, University of Washington, United States
Research Article
Cite this article as: eLife 2020;9:e57659 doi: 10.7554/eLife.57659
6 figures, 2 tables, 18 data sets and 2 additional files

Figures

Figure 1 with 1 supplement
De novo design of protein nanoparticles tailored for multivalent antigen presentation.

(a) Computational docking of monomeric repeat proteins into C3-symmetric trimers using the RPX method. (b) Selection of trimers for design based on close geometric match between their N termini (blue spheres) and C termini (red spheres) of a viral antigen (green, BG505 SOSIP shown for illustration). (c) Design of two-component nanoparticles incorporating a fusion component (cyan) and assembly component (gray). (d) Nanoparticle assembled with antigen-fused trimeric component yields multivalent antigen-displaying nanoparticle.

Figure 1—figure supplement 1
Computational docking and design of trimers for fusion to a specific viral glycoprotein.

Design models for C3-symmetric trimers (gray) with labeled N termini (blue spheres) screened against the BG505 SOSIP trimeric glycoprotein subunit gp41 (cyan) with C-terminal residues labeled (red spheres). 

Figure 2 with 7 supplements
Biophysical characterization of antigen-tailored trimers and nanoparticles.

Top rows, design models. Middle rows, SEC chromatograms and calculated molecular weights from SEC-MALS. Bottom rows, comparisons between experimental SAXS data and scattering profiles calculated from design models. (a) 1na0C3_2. (b) 3ltjC3_1v2. (c) 3ltjC3_11. (d) HR04C3_5v2. (e) T33_dn2. (f) T33_dn10. (g) O43_dn18. (h) I53_dn5.

Figure 2—source data 1

Biophysical properties of designed trimers and two-component nanoparticles.

Experimentally-measured data (exp) is compared to predicted design data (model). Molecular weights (MW) were obtained using the ASTRA software. Rg and Dmax calculations performed in Scatter3 SAXS analysis software with the determined qmax values. X values computed from the FoXS online SAXS web server between the designed model and the experimental scattering data.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data1-v1.docx
Figure 2—source data 2

1na0C3_2 SEC-MALS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data2-v1.txt
Figure 2—source data 3

3ltjC3_1v2 SEC-MALS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data3-v1.txt
Figure 2—source data 4

3ltjC3_11 SEC-MALS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data4-v1.txt
Figure 2—source data 5

HR04C3_5v2 SEC-MALS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data5-v1.txt
Figure 2—source data 6

1na0C3_2 SAXS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data6-v1.txt
Figure 2—source data 7

3ltjC3_1v2 SAXS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data7-v1.txt
Figure 2—source data 8

3ltjC3_11 SAXS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data8-v1.txt
Figure 2—source data 9

HR04_5v2 SAXS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data9-v1.txt
Figure 2—source data 10

T33_dn2 SEC-MALS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data10-v1.txt
Figure 2—source data 11

T33_dn10 SEC-MALS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data11-v1.txt
Figure 2—source data 12

O43_dn18 SEC-MALS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data12-v1.txt
Figure 2—source data 13

I53_dn5 SEC-MALS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data13-v1.txt
Figure 2—source data 14

T33_dn2 SAXS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data14-v1.txt
Figure 2—source data 15

T33_dn10 SAXS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data15-v1.txt
Figure 2—source data 16

O43_dn18 SAXS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data16-v1.txt
Figure 2—source data 17

I53_dn5 SAXS.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-data17-v1.txt
Figure 2—figure supplement 1
SEC-MALS chromatograms for designed trimers occupying an off-target oligomeric state.

Predominant oligomeric species for each design were collected by fractionation from initial SEC purification, and fourteen chromatograms are presented here from a subsequent round of high-performance SEC-MALS using a Superdex 200 column. *Chromatogram obtained using a Superdex 75 10/300 GL column.

Figure 2—figure supplement 1—source data 1

SEC-MALS data for off-target designed trimers.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-figsupp1-data1-v1.docx
Figure 2—figure supplement 2
SEC chromatograms for designed trimers with off-target retention volumes after Ni2+ IMAC.

Primary SEC chromatograms obtained from a Superdex 200 column for soluble proteins after purification by Ni2+ IMAC. Designs presented here formed off-target or polydisperse assemblies based on retention volume.

Figure 2—figure supplement 3
Comparison between the experimentally determined crystal structures and corresponding models of two designed trimers.

Left, design models (gray) and crystal structures (gray) superposed indicating resolution of structure (res.) and backbone r.m.s.d. between structure and design model. Right, magnified view of the de novo designed interface and side chain packing.

Figure 2—figure supplement 3—source data 1

Crystallography data collection and refinement statistics for designed trimers 1na0C3_2 and 3ltjC3_1v2.

Statistics for the highest-resolution shell are shown in parentheses.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig2-figsupp3-data1-v1.docx
Figure 2—figure supplement 4
SDS-PAGE of bicistronically-expressed designed nanoparticles eluted from Ni2+ IMAC.

For each designed nanoparticle: Left - protein standard (Precision Plus Dual Xtra, Bio-Rad). Right - labeled bands corresponding to the expected size of each component.

Figure 2—figure supplement 5
SEC profiles for two-component nanoparticles with off-target retention volumes after Ni2+ IMAC.

Primary SEC chromatograms obtained from a Superose six column for soluble proteins directly after purification by Ni2+ IMAC. Designs presented here formed off-target or polydisperse assemblies based on retention volume.

Figure 2—figure supplement 6
Biophysical characterization of T33_dn5.

Left - designed model. Middle - SEC chromatograms and calculated molecular weights from SEC-MALS. Right - comparisons between experimental SAXS data and scattering profile computed from the design model.

Figure 2—figure supplement 7
In vitro assembly of I53_dn5.

SEC chromatograms of individual components I53_dn5A (pentameric assembly component, cyan) and I53_dn5B (trimeric fusion component, gray), and nanoparticle purified from equimolar assembly run on a Superose 6 10/300 GL column.

NS-EM analysis of antigen-tailored nanoparticles.

From left to right: designed trimers incorporated in each designed nanoparticle, nanoparticle design models fit into NS-EM density (views shown down each component axis of symmetry), designed nanoparticle 2D class-averages, raw electron micrographs of designed nanoparticles. (a) T33_dn2. (b) T33_dn5. (c) T33_dn10. (d) O43_dn18. (e) I53_dn5.

Figure 4 with 1 supplement
Cryo-EM analysis of antigen-tailored nanoparticles.

From left to right: cryo-EM maps with refined nanoparticle design models fit into electron density, view of designed nanoparticle interface region fit into cryo-EM density with indicated resolution (res.), designed nanoparticle 2D class-averages, raw cryo-EM micrographs of designed nanoparticles. (a) T33_dn10. (b) O43_dn18. (c) I53_dn5.

Figure 4—source data 1

Cryo-EM data acquisition metrics for designed nanoparticles T33_dn10, O43_dn18, and I53_dn5.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig4-data1-v1.docx
Figure 4—source data 2

Cryo-EM model building and refinement statistics for designed nanoparticles T33_dn10, O43_dn18, and I53_dn5.

https://cdn.elifesciences.org/articles/57659/elife-57659-fig4-data2-v1.docx
Figure 4—figure supplement 1
Cryo-EM data processing workflow.

Statistics are presented for (a) T33_dn10, (b) O43_dn18, and (c) I53_dn5.

Figure 5 with 2 supplements
NS-EM analysis of BG505 SOSIP-displaying nanoparticles.

From left to right: BG505 SOSIP-displaying nanoparticle models fit into NS-EM density, 2D class-averages, raw NS-EM micrographs of assembled BG505 SOSIP-displaying nanoparticles. (a) BG505 SOSIP–T33_dn2. (b) BG505 SOSIP–T33_dn10. (c) BG505 SOSIP–I53_dn5.

Figure 5—figure supplement 1
Structural and antigenic characterization of DS-Cav1–I53_dn5.

Top panel: ELISA using anti-DS-Cav1 antibodies D25, Motavizumab (Mota), AM14, or negative control CR6261 added to DS-Cav1 trimer with foldon or antigen-displaying nanoparticle DS-Cav1–I53_dn5. Bottom panel: SEC chromatogram of DS-Cav1–I53_dn5 on a Superose six column and NS-EM field image and 2D class averages for DS-Cav1–I53_dn5.

Figure 5—figure supplement 2
Structural and antigenic characterization of HA–I53_dn5.

Top panel: Octet bio-layer interferometry using plate-coated head-directed mAb 5J8 for antigen capture, and subsequent stem-directed mAb CR6261 addition to both antigen-fused trimeric component HA–I53_dn5B and antigen-displaying nanoparticle HA–I53_dn5. Bottom panel: SEC chromatogram for nanoparticle HA–I53_dn5 compared to trimer HA–I53_dn5B on a Sephacryl S-500 column, and NS-EM field image and 2D class averages for HA–I53_dn5.

BG505 SOSIP epitope accessibility compared between tetrahedral and icosahedral presentation geometries.

(a) NS-EM micrographs of BG505 SOSIP–T33_dn2A with and without VRC01 Fab bound, 2D class averages, and models fit into NS-EM density. (b) Representative sensorgrams of indicated proteins binding to anti-Env mAbs. (c) Relative accessibility of epitopes on BG505 SOSIP–T33_dn2 nanoparticles and BG505 SOSIP–I53-50 nanoparticles as determined by mAb binding (top). Ratio of moles of macromolecules are means of 2–4 experimental replicates. Epitopes mapped onto BG505 SOSIP are presented on models of T33_dn2 and I53-50 (bottom). Wheat, antigen-fused trimeric component; purple, assembly component; gray, neighboring BG505 SOSIP trimers on the nanoparticle surface.

Tables

Table 1
Summary of the experimental characterization for designed trimers and two-component nanoparticles.

1na0C3_2 and 3ltjC3_1v2 structures determined by X-ray crystallography and T33_dn10, O43_dn18, and I53_dn5 structures determined by cryo-EM.

DesignTargeted AntigensExperimental Molecular
Weight (kDa)
Target Molecular
Weight (kDa)
SAXS X valueResolution, backbone r.m.s.d.
structure (Å, Å)
1na0C3_2HA, SOSIP, DS-Cav148451.42.6, 1.4
3ltjC3_1v2SOSIP, DS-Cav156631.12.3, 0.8
3ltjC3_11SOSIP, DS-Cav150661.6--
HR04C3_5v2SOSIP71691.5--
T33_dn2HA, SOSIP, DS-Cav13973454.8--
T33_dn5HA, SOSIP, DS-Cav14224221.7--
T33_dn10HA, SOSIP, DS-Cav15465562.33.9, 0.65
O43_dn18HA,SOSIP, DS-Cav18108762.94.5, 0.98
I53_dn5HA, SOSIP, DS-Cav1200019601.25.3, 1.30
Table 1—source data 1

Summary of the experimental characterization for designed trimers and two-component nanoparticles.

https://cdn.elifesciences.org/articles/57659/elife-57659-table1-data1-v1.docx
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Software, algorithmRPX MethodPMID:28338692Symmetric docking and scoring protocol
Software, algorithmSic_axlePMID:30849373Protein structure alignment protocol
Software, algorithmRosetta Macromolecular
Modeling Suite
PMID:28430426RRID:SCR_015701Version 3
Software, algorithmRelionPMID:23000701RRID:SCR_016274Cryo-EM structure
determination software
Strain, strain background (E. coli)BL21New England BiolabsCat. #:C2527HCompetent T7 expression strain
Strain, strain background (E. coli)Lemo21New England BiolabsCat. #:C2527HCompetent T7 expression strain
Strain, strain background (E. coli)HEK293FPMID:26779721RRID:CVCL_6642Suspension-based cells for high yield expression of recombinant proteins
Chemical compound, drugIPTGSigmaCat. #:I6758Induces protein expression through T7 promoter
Chemical compound, drugKanamycinSigmaCat. #:K1377Antibiotic
Chemical compound, drugCarbenicillinSigmaCat. #:C1389Antibiotic
Chemical compound, drugExpifectamineThermoFisherCat. #:A38915Transfection reagent
Chemical compound, drugPolyethyleniminePolysciences IncCat. #:23966Transfection reagent
Recombinant DNA reagentpET21b(+)GenscriptAddgene Cat. #:69741–3Bacterial expression vector
Recombinant DNA reagentpET28b(+)Gen9Addgene Cat. #:69865–3Bacterial expression vector
Recombinant DNA reagentpPPI4Progenics Pharmaceuticals Inc
PMID:10623724
Mammalian secretion vector, containing codon-optimized stabilized gp140
Recombinant DNA reagentCMV/RPMID:15994776Mammalian secretion vector, containing CMV
enhancer/promoter with HTLV-1 R region
AntibodyPGT145 human monoclonalPMID:21849977RRID:AB_2491054anti-HIV-1 Env (anti-Fc immobilization level of 320 ± 1.5 RU)
AntibodyPGT122 human
monoclonal
PMID:21849977RRID:AB_2491042anti-HIV-1 Env (anti-Fc immobilization level of 320 ± 1.5 RU)
Antibody2G12 human monoclonalPMID:8551569RRID:AB_2819235anti-HIV-1 Env (anti-Fc immobilization level of 320 ± 1.5 RU)
AntibodyVRC01 human monoclonalPMID:20616233RRID:AB_2491019anti-HIV-1 Env (anti-Fc immobilization level of 320 ± 1.5 RU)
AntibodyACS202 human monoclonalPMID:27841852anti-HIV-1 Env (anti-Fc immobilization level of 320 ± 1.5 RU)
AntibodyVRC34 human monoclonalPMID:27174988RRID:AB_2819228anti-HIV-1 Env (anti-Fc immobilization level of 320 ± 1.5 RU)
AntibodyPGT151 human monoclonalPMID:24768347anti-HIV-1 Env (anti-Fc immobilization level of 320 ± 1.5 RU)
Antibody3BC315 human monoclonalPMID:22826297anti-HIV-1 Env (anti-Fc immobilization level of 320 ± 1.5 RU)
Antibody11B rabbit monoclonalPMID:27545891anti-HIV-1 Env (anti-Fc immobilization level of 320 ± 1.5 RU)
Antibody12N rabbit monoclonalPMID:27545891anti-HIV-1 Env (anti-Fc immobilization level of 320 ± 1.5 RU)
Antibody5J8 human monoclonalPMID:21849447anti-HA (20 μg/mL)
AntibodyCR6261 human monoclonalPMID:19079604anti-HA (20 μg/mL)
AntibodyD25 human monoclonalPMID:24179220anti-RSV F (1 pg/mL - 10 μg/mL)
AntibodyMotavizumab mouse-human chimeric monoclonalPMID:20065632anti-RSV F (1 pg/mL - 10 μg/mL)
AntibodyAM14 human monoclonalPMID:26161532anti-RSV F (1 pg/mL - 10 μg/mL)

Data availability

Diffraction data have been deposited in the PDB under accession codes 6V8E and 6VEH. Cyro-EM structures have been deposited in the PDB under accession codes 6VFH, 6VFI, and 6VFJ. Electron density maps have been deposited in the EMDB with numbers 21162, 21163, 21164, 21165, 21166, 21167, 21168, 21169, 21170, 21171, 21172, 21173, and 21174. All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2, 4 and 6 specifically.

The following data sets were generated
  1. 1
    RCSB Protein Data Bank
    1. B Sankaran
    2. G Ueda
    3. PH Zwart
    4. D Baker
    (2020)
    ID 6V8E. Tailored Design of Protein Nanoparticle Scaffolds for Viral Glycoprotein Immunogens.
  2. 2
    RCSB Protein Data Bank
    1. MJ Bick
    2. G Ueda
    3. D Baker
    (2020)
    ID 6VEH. Computationally designed C3-symmetric homotrimer from HEAT repeat protein.
  3. 3
    Electron Microscopy Data Bank
    1. A Antanasijevic
    2. G Ueda
    3. D Baker
    4. AB Ward
    (2020)
    ID 21162. De novo designed tetrahedral nanoparticle T33_dn2.
  4. 4
    Electron Microscopy Data Bank
    1. A Antanasijevic
    2. G Ueda
    3. D Baker
    4. AB Ward
    (2020)
    ID 21163. De novo designed tetrahedral nanoparticle T33_dn5.
  5. 5
    Electron Microscopy Data Bank
    1. A Antanasijevic
    2. G Ueda
    3. D Baker
    4. AB Ward
    (2020)
    ID 21164. De novo designed tetrahedral nanoparticle T33_dn10.
  6. 6
    Electron Microscopy Data Bank
    1. A Antanasijevic
    2. G Ueda
    3. D Baker
    4. AB Ward
    (2020)
    ID 21165. De novo designed octahedral nanoparticle O43_dn18.
  7. 7
    Electron Microscopy Data Bank
    1. A Antanasijevic
    2. G Ueda
    3. D Baker
    4. AB Ward
    (2020)
    ID 21166. De novo designed icosahedral nanoparticle I53_dn5.
  8. 8
    Electron Microscopy Data Bank
    1. A Antanasijevic
    2. G Ueda
    3. D Baker
    4. AB Ward
    (2020)
    ID 21167. BG505-SOSIP-T33_dn2A nanoparticle fusion component.
  9. 9
    Electron Microscopy Data Bank
    1. A Antanasijevic
    2. G Ueda
    3. D Baker
    4. AB Ward
    (2020)
    ID 21168. BG505-SOSIP-T33_dn2A nanoparticle fusion component in complex with VRC01-Fab.
  10. 10
    Electron Microscopy Data Bank
    1. A Antanasijevic
    2. G Ueda
    3. D Baker
    4. AB Ward
    (2020)
    ID 21169. De novo designed tetrahedral nanoparticle T33_dn2 presenting BG505-SOSIP.
  11. 11
    Electron Microscopy Data Bank
    1. A Antanasijevic
    2. G Ueda
    3. D Baker
    4. AB Ward
    (2020)
    ID 21170. Tetrahedral nanoparticle T33_dn10 presenting BG505-SOSIP.
  12. 12
    Electron Microscopy Data Bank
    1. A Antanasijevic
    2. G Ueda
    3. D Baker
    4. AB Ward
    (2020)
    ID 21171. Icosahedral Nanoparticle I53_dn5 presenting BG505-SOSIP.
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18

Additional files

Supplementary file 1

Sequences for all designed trimers, homo-oligomers, two-component nanoparticles, and antigen-fused components.

(A) Amino acid sequences for all designed trimers and de novo homo-oligomers used for two-component nanoparticle design. Sequences include initiating methionines and His6-tags. Designed trimers that expressed solubly are denoted in bold, and experimental methods used for characterization are included in parentheses. *Components from previously described designed homo-oligomers in Fallas et al., 2017 or the Protein Data Bank (PDB ID). (B) Amino acid sequences for all designed two-component nanoparticles. Sequences include initiating methionines and His6-tags. Designs that expressed solubly and co-eluted from IMAC are denoted in bold. Input oligomers from (A) are included in parentheses. (C) Amino acid sequences for all antigen-fused trimeric nanoparticle components. Sequences include initiating methionines and signal peptides.

https://cdn.elifesciences.org/articles/57659/elife-57659-supp1-v1.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/57659/elife-57659-transrepform-v1.docx

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