Author response: Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens (2020)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Multivalent presentation of viral glycoproteins can substantially increase the elicitation of antigen-specific antibodies. To enable a new generation of anti-viral vaccines, we designed self-assembling protein nanoparticles with geometries tailored to present the ectodomains of influenza, HIV, and RSV viral glycoprotein trimers. We first de novo designed trimers tailored for antigen fusion, featuring N-terminal helices positioned to match the C termini of the viral glycoproteins. Trimers that experimentally adopted their designed configurations were incorporated as components of tetrahedral, octahedral, and icosahedral nanoparticles, which were characterized by cryo-electron microscopy and assessed for their ability to present viral glycoproteins. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles presented antigenically intact prefusion HIV-1 Env, influenza hemagglutinin, and RSV F trimers in the predicted geometries. This work demonstrates that antigen-displaying protein nanoparticles can be designed from scratch, and provides a systematic way to investigate the influence of antigen presentation geometry on the immune response to vaccination. eLife digest Vaccines train the immune system to recognize a specific virus or bacterium so that the body can be better prepared against these harmful agents. To do so, many vaccines contain viral molecules called glycoproteins, which are specific to each type of virus. Glycoproteins that sit at the surface of the virus can act as 'keys' that recognize and unlock the cells of certain organisms, leading to viral infection. To ensure a stronger immune response, glycoproteins in vaccines are often arranged on a protein scaffold which can mimic the shape of the virus of interest and trigger a strong immune response. Many scaffolds, however, are currently made from natural proteins which cannot always display viral glycoproteins. Here, Ueda, Antanasijevic et al. developed a method that allows for the design of artificial proteins which can serve as scaffold for viral glycoproteins. This approach was tested using three viruses: influenza, HIV, and RSV – a virus responsible for bronchiolitis. The experiments showed that in each case, the relevant viral glycoproteins could attach themselves to the scaffold. These structures could then assemble themselves into vaccine particles with predicted geometrical shapes, which mimicked the virus and maximized the response from the immune system. Designing artificial scaffold for viral glycoproteins gives greater control over vaccine design, allowing scientists to manipulate the shape of vaccine particles and test the impact on the immune response. Ultimately, the approach developed by Ueda, Antanasijevic et al. could lead to vaccines that are more efficient and protective, including against viruses for which there is currently no suitable scaffold. Introduction Multivalent antigen presentation, in which antigens are presented to the immune system in a repetitive array, has been demonstrated to increase the potency of humoral immune responses (Bennett et al., 2015; Snapper, 2018). This has been attributed to increased cross-linking of antigen-specific B cell receptors at the cell surface and modulation of immunogen trafficking to and within lymph nodes (Irvine et al., 2013; Tokatlian et al., 2019). An ongoing challenge has been to develop multimerization scaffolds capable of presenting complex oligomeric or engineered antigens (Sanders and Moore, 2017; Jardine et al., 2013; McLellan et al., 2013a), as these can be difficult to stably incorporate into non-protein-based nanomaterials (e.g. liposomes, polymers, transition metals and their oxides). Epitope accessibility, proper folding of the antigen, and stability are also important considerations in any strategy for antigen presentation. Several reports have utilized non-viral, naturally occurring protein scaffolds, such as self-assembling ferritin (Kanekiyo et al., 2013; Sliepen et al., 2015; Darricarrère et al., 2018), lumazine synthase (Sanders and Moore, 2017; Abbott et al., 2018), or encapsulin (Kanekiyo et al., 2015) nanoparticles, to present a variety of complex oligomeric or engineered antigens. These studies have illustrated the advantages of using self-assembling proteins as scaffolds for antigen presentation (López-Sagaseta et al., 2016; Kanekiyo et al., 2019), including enhanced immunogenicity and seamless integration of antigen and scaffold through genetic fusion. More recently, computationally designed one- and two-component protein nanoparticles (Hsia et al., 2016; King et al., 2014; Bale et al., 2016) have been used to present complex oligomeric antigens, conferring additional advantages such as high stability, robust assembly, ease of production and purification, and increased potency upon immunization (Marcandalli et al., 2019; Brouwer et al., 2019). The ability to predictively explore new structural space makes designed proteins (Parmeggiani et al., 2015; Brunette et al., 2015) attractive scaffolds for multivalent antigen presentation. In our previous work with computationally designed nanoparticle immunogens (Marcandalli et al., 2019; Brouwer et al., 2019), the nanoparticles were generated from naturally occurring oligomeric proteins without initial consideration of geometric compatibility for antigen presentation. A more comprehensive solution would be to de novo design nanoparticles which present complex antigens of interest. For homo-oligomeric class I viral fusion proteins, a large group that includes many important vaccine antigens (Harrison, 2015), a close geometric match between the C termini of the antigen and the N termini of a designed nanoparticle component would enable multivalent presentation without structural distortion near the glycoprotein base, and potentially allow for better retention of antigenic epitopes relevant to protection. More generally, precise control of antigen presentation geometry through de novo nanoparticle design would enable systematic investigation of the structural determinants of immunogenicity. Results De novo design of protein nanoparticles tailored for multivalent antigen presentation We sought to develop a general computational method for de novo designing protein nanoparticles with geometries tailored to present antigens of interest, focusing specifically on the prefusion conformations of the trimeric viral glycoproteins HIV-1 Env (BG505 SOSIP) (Wang et al., 2017; Sanders et al., 2013), influenza hemagglutinin (H1 HA) (Kadam et al., 2017), and respiratory syncytial virus (RSV) F (DS-Cav1) (McLellan et al., 2013a). To make the antigen-tailored nanoparticle design problem computationally tractable, we employed a two-step design approach (Figure 1). In the first step, we de novo designed antigen-tailored trimers, featuring N termini geometrically matched to the C termini of the viral glycoproteins. In the second step, we generated tetrahedral, octahedral, and icosahedral two-component nanoparticles by designing secondary interfaces between a designed trimer (fusion component) and a de novo homo-oligomer (assembly component) (Fallas et al., 2017). This design approach yielded nanoparticles tailored to present 4, 8, or 20 copies of the viral glycoproteins in defined geometries (Figure 1d). Sequences for all designed trimers and homo-oligomers, two-component nanoparticles, and antigen-fused components in this study can be found in Supplementary file 1A, B, and C, respectively. Details on each step of the design approach are described in the following sections. Figure 1 with 1 supplement see all Download asset Open asset 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. Computational design of trimers tailored for fusion to specific viral glycoproteins We chose to design our antigen-tailored trimers from monomeric repeat proteins composed of rigidly packed 20- to 50-residue tandem repeat units (Parmeggiani et al., 2015; Brunette et al., 2015; Urvoas et al., 2010; Kajander et al., 2007; Main et al., 2003), as their high stability and tunable length (through variation of repeat number) are desirable properties for the design of protein-based nanomaterials. These structurally diverse alpha-helical repeat proteins featured three to six repeat modules and total lengths between 119 and 279 residues. They were docked into C3-symmetric trimers using our RPX docking method, which identifies configurations likely to accommodate favorable side chain packing at the de novo designed interface (Fallas et al., 2017). To identify trimeric configurations with N termini compatible for fusion to the C termini of the three viral glycoproteins, docks with an RPX score above 5.0 were screened using the sic_axle protocol (Marcandalli et al., 2019). Geometrically compatible docks (non-clashing termini separation distances of 15 Å or less) were subjected to full Rosetta C3-symmetric interface design and filtering (see Materials and Methods), and twenty-three designs were selected for experimental characterization (Figure 1—figure supplement 1). Structural characterization of designed trimers Synthetic genes encoding each of the designed trimers were expressed in E. coli and purified from lysates by Ni2+ immobilized metal affinity chromatography (Ni2+ IMAC) followed by size-exclusion chromatography (SEC). Twenty-two designs were found to express in the soluble fraction, and nine formed the intended trimeric oligomerization state as assessed by SEC in tandem with multi-angle light scattering (SEC-MALS; examples in Figure 2 top panel, second row; SEC-MALS chromatograms for the remaining designs are in Figure 2—figure supplement 2 and data in Figure 2—figure supplement 1—source data 1; SEC chromatograms for remaining designs with off-target retention volumes are in Figure 2—figure supplement 2). Four of the designs that were trimeric and expressed in high yield, 1na0C3_2, 3ltjC3_1v2, 3ltjC3_11, and HR04C3_5v2, were selected for solution small angle X-ray scattering (SAXS) experiments. The proteins exhibited scattering profiles very similar to those computed from the corresponding design models, suggesting similar supramolecular configuration (Figure 2 top panel, third row; metrics in Table 1 and Figure 2—source data 1). These four trimers were derived from three distinct designed helical repeat proteins from TPR, HEAT, or de novo topological families (1na0, 3ltj, and HR04, see Materials and Methods) (Brunette et al., 2015; Urvoas et al., 2010; Main et al., 2003). Crystals were obtained for the two designs 1na0C3_2 and 3ltjC3_1v2. Structures were determined at resolutions of 2.6 and 2.3 Å, revealing a backbone root mean square deviation (r.m.s.d.) between the design model and structure of 1.4 and 0.8 Å, respectively (Figure 2—figure supplement 3, and Figure 2—figure supplement 3—source data 1, crystallization conditions, structure metrics, and structure-to-model comparisons are described in Materials and Methods). The structures confirmed in both cases that the designed proteins adopt the intended trimeric configurations, and that most of the atomic details at the de novo designed interfaces are recapitulated. Figure 2 with 7 supplements see all Download asset Open asset 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 Download 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 Download 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 Download 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 Download 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 Download 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 Download 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 Download 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 Download 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 Download 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 Download 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 Download 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 Download 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 Download 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 Download 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 Download 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 Download 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 Download elife-57659-fig2-data17-v1.txt 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, 1.43ltjC3_1v2SOSIP, DS-Cav156631.12.3, 0.83ltjC3_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, 0.65O43_dn18HA,SOSIP, DS-Cav18108762.94.5, 0.98I53_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 Download elife-57659-table1-data1-v1.docx Computational design of two-component nanoparticles incorporating designed trimers As secondary assembly components were required to design our antigen-tailored nanoparticles, validated trimers were docked pairwise with de novo designed symmetric homo-oligomers (Fallas et al., 2017) to generate tetrahedral, octahedral, and icosahedral nanoparticle configurations using the TCdock program (King et al., 2014; Bale et al., 2016). To increase the probability of generating icosahedra which confer the highest valency among the targeted symmetries, three naturally occurring homopentamers were also included in the docking calculations (PDB IDs 2JFB, 2OBX, and 2B98). Analogously to the designed trimers, nanoparticle docks were scored and ranked using the RPX method (Fallas et al., 2017) to identify configurations likely to accommodate favorable side chain packing at a secondary de novo designed interface. High-ranking and non-redundant nanoparticle configurations featuring outward-facing N termini for antigen presentation were selected for Rosetta interface design (King et al., 2014; Bale et al., 2016). Fifty-three nanoparticle designs across all three targeted symmetries that exhibited the best interface metrics were selected for experimental characterization (see Materials and Methods). The nomenclature for the eleven tetrahedra, twenty-one octahedra, and twenty-one icosahedra indicate the symmetry of the nanoparticle (T, O, or I), the oligomeric state of the first component (A) and second component (B) used in each design, the letters "dn" reflecting the de novo nature of the input oligomers, and the rank by RPX score from the docking stage (e.g., "I53_dn5" indicates an icosahedral nanoparticle constructed from a pentameric and trimeric component, ranked 5th in RPX-scoring for the two input oligomers). Synthetic genes encoding each of the two-component nanoparticles were obtained with one of the components fused to a His6-tag, and the designs were purified using Ni2+ IMAC (see Materials and methods). Pairs of proteins at the expected molecular weights were found to co-elute by SDS-PAGE for twenty-four of the designs, consistent with spontaneous assembly of the nanoparticles followed by pulldown His6-tagged component (featured co-eluting designs are presented in Figure 2—figure supplement 4). SEC chromatograms revealed that nineteen designs did not form assemblies of the expected size or that the resulting assemblies were heterogeneous (Figure 2—figure supplement 5). Five designs comprising a panel of unique geometric configurations, T33_dn2, T33_dn5, T33_dn10, O43_dn18, and I53_dn5, ran as monodisperse particles of the predicted molecular mass by SEC-MALS and were further investigated by SAXS. The experimental solution scattering curves closely matched the scattering curves computed from the design models (Schneidman-Duhovny et al., 2010) for all five designs (Figure 2, bottom panel and Figure 2—figure supplement 6; metrics in Table 1 and Figure 2—source data 1, bottom five designs). Due to its high valency and production yield, we selected the I53_dn5 nanoparticle to investigate the capacity of its two components to be separately produced and assembled in vitro. The two components of I53_dn5 were re-cloned, expressed, and separately purified (pentameric "I53_dn5A" with His6-tag and trimeric "I53_dn5B"). Nanoparticle assembly appeared to be complete within minutes after equimolar mixing (Figure 2—figure supplement 7). This capability is noteworthy as it enables production of each component independently, even from different host systems, which provides more flexibility in nanoparticle manufacturing. In vitro assembly also confers more control over nanoparticle assembly and composition, for example by assembling with a mixture of components fused to different antigens (Boyoglu-Barnum et al., 2020). Structural characterization of designed two-component nanoparticles The five SAXS-validated nanoparticles were structurally characterized using negative stain electron microscopy (NS-EM) (Lee and Gui, 2016; Ozorowski et al., 2018). 2,000–5000 particles were manually picked from the electron micrographs acquired for each designed nanoparticle and classified in 2D using the Iterative MSA/MRA algorithm (see Materials and Methods). 3D classification and refinement steps were performed in Relion/3.0 (Zivanov et al., 2018). Analysis of the NS-EM data confirmed high sample homogeneity for all five nanoparticle designs as evident from the micrographs and 2D class-averages (Figure 3). While some free nanoparticle components were detected in the T33_dn5 sample, suggesting a certain propensity towards disassembly, analysis of the reconstructed 3D maps revealed that all five nanoparticles assemble as predicted by the design models, at least to the resolution limits of NS-EM. Figure 3 Download asset Open asset 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. In order to obtain higher-resolution information, three designs, T33_dn10, O43_dn18, and I53_dn5, representing one nanoparticle from each targeted symmetry (T, O, I), were subjected to cryo-electron microscopy (cryo-EM). Cryo-EM data acquisition was performed as described in the Materials and Methods section and data acquisition statistics are displayed in Figure 4—source data 1. The data processing workflow is presented in Figure 4—figure supplement 1. Appropriate symmetry (T, O, and I for T33_dn10, O43_dn18, and I53_dn5, respectively) was applied during 3D classification and refinement and maps were post-processed in Relion/3.0 (Zivanov et al., 2018). The final resolutions of the reconstructed maps for the T33_dn10, O43_dn18, and I53_dn5 nanoparticles were 3.9, 4.5, and 5.3 Å, respectively. Some structural heterogeneity was observed in the cryo-EM data, particularly in the case of I53_dn5. In 2D classification results we generated particle projection averages that from to shape (Figure 4—figure supplement some of is of flexibility in T33_dn10 and O43_dn18, in with the final resolution for these nanoparticles. Nanoparticle design models were into the corresponding maps by of Rosetta refinement (Wang et al., 2016) and refinement in and to generate the final model statistics are shown in Figure 4—source data cryo-EM maps for T33_dn10, O43_dn18, and I53_dn5 and models are in Figure the structures with the corresponding Rosetta design models. r.m.s.d. values for the of a of component A and component were and Å for T33_dn10, O43_dn18, and I53_dn5, respectively 1). Figure 4 with 1 supplement see all Download asset Open asset Cryo-EM analysis of antigen-tailored nanoparticles. From left to right: cryo-EM maps with nanoparticle design models fit into electron of designed nanoparticle interface fit into cryo-EM density with resolution 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. Download Figure 4—source data 2 Cryo-EM model and refinement statistics for designed nanoparticles T33_dn10, O43_dn18, and I53_dn5. Download of viral nanoparticles To explore the capability of the designed nanoparticles to present viral glycoproteins, we produced their trimeric fusion components to a of the BG505 SOSIP Synthetic genes for BG505 SOSIP fused to the N termini of and (BG505 BG505 and BG505 were into The fusion proteins were then purified using a of chromatography and The corresponding assembly component for each nanoparticle was produced in E. and in vitro assembly were performed as equimolar of the two components nanoparticles were purified by SEC and by NS-EM to particle assembly and particles were manually picked and used to 2D classification and 3D in (Zivanov et al., 2018). for the BG505 nanoparticles fit into their reconstructed 3D maps are displayed in Figure 5 BG505 SOSIP trimers are in 2D class-averages and reconstructed 3D the trimers the corresponding nanoparticle in the three likely to the between the BG505 SOSIP trimer and the fusion The self-assembling of the antigen-fused T33_dn2, T33_dn10, and I53_dn5 nanoparticles were very similar to the NS-EM maps of the nanoparticles least to the resolution limits of that fusion of the BG505 SOSIP trimer did not any structural to the nanoparticle components were detected in raw micrographs of BG505 some of This is by stability data in a BG505 demonstrated to and et al., 2020). Figure 5 with 2 supplements see all Download asset Open asset NS-EM analysis of BG505 nanoparticles. From left to right: BG505 nanoparticle models fit into NS-EM 2D class-averages, raw NS-EM micrographs of assembled BG505 nanoparticles. (a) BG505 (b) BG505 (c) BG505 To further the capability of the designed nanoparticles to present viral glycoproteins, we characterized the structures and antigenic profiles of I53_dn5 fused to the prefusion influenza and RSV F glycoproteins and were generated with each glycoprotein to the N of the trimeric fusion component, and the proteins were from cells and purified by Ni2+ The fusion proteins were with equimolar pentameric for or and of see Materials and Methods) for and the assembly purified by For both the of the in the expected for assembled nanoparticles, and NS-EM analysis showed of I53_dn5 nanoparticles with from the surface (Figure supplement 1 and 2). In both there was variation in the suggesting some flexibility between the glycoproteins and the scaffold. The to I53_dn5 likely for the observed flexibility and of the glycoprotein trimer in class averages (Figure supplement 1, bottom was no engineered between the glycoprotein and fusion component in the case of and more defined density was observed in the class averages (Figure supplement 2, bottom To the presented glycoproteins were we their with The nanoparticle was found by an to the RSV (McLellan et al., and et al., 2015) to soluble trimer with (McLellan et al., 2013a), that the F protein is presented in the prefusion on the nanoparticle (Figure 4—figure supplement 1, binding experiments with and et al., et al., showed that both the nanoparticle and the trimer presented the and with (Figure supplement 2,