A single immunization with spike-functionalized ferritin vaccines elicits neutralizing antibody responses against SARS-CoV-2 in mice

Development of a safe and effective SARS-CoV-2 vaccine is a public health priority. We designed subunit vaccine candidates using self-assembling ferritin nanoparticles displaying one of two multimerized SARS-CoV-2 spikes: full-length ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion (SΔC-Fer). Ferritin is an attractive nanoparticle platform for production of vaccines and ferritin-based vaccines have been investigated in humans in two separate clinical trials. We confirmed proper folding and antigenicity of spike on the surface of ferritin by cryo-EM and binding to conformation-specific monoclonal antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients. Additionally, a single dose of SΔC-Fer elicited significantly higher neutralizing responses as compared to immunization with the spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with SΔC-Fer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentation of SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy for single-dose vaccination against COVID-19.

. CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 28, 2020. . https://doi.org/10.1101/2020.08.28.272518 doi: bioRxiv preprint Figure S2. Size-exclusion chromatography multi-angle light scattering molecular weight determination of spike ferritin nanoparticles and analysis of other antigens. (A) Light scattering analysis of the S-Fer and SΔC-Fer particles on the SRT SEC-1000 analytical column indicate that particles do not aggregate following a freeze-thaw cycle. Glycerol (10%) was added to particle samples prior to snapfreezing. (B) Molecular weight calculation for S-Fer and SΔC-Fer determined by SEC-MALS was performed with ASTRA software using light scattering and refractive index signals for the particles. The average calculated molecular weight obtained from two independent protein preparations for each particle (S-Fer and SΔC-Fer) is shown. The expected molecular weight was determined by the amino acid sequence for the individual S-Fer and SΔC-Fer protomers and multiplied by 24 to account for the number of protomers in a particle. The expected mass does not account for glycosylation; each protomer contains ~20 predicted N-linked glycans which could add up to 1 MDa to the mass of the particle (58). Discrepancies in calculated and expected molecular weights could in part be due to incomplete glycosylation. The plots show a representative curve from each analysis. The colored traces correspond to the left y-axis which shows the Rayleigh ratio, a measure of light scattering. The black line on each peak is the calculated molecular weight, plotted on the right y-axis, as a function of particle elution. This demonstrates that the molecular weight calculation is not subject to variations resulting from artifacts in the eluted peak. (C) SEC-MALS traces for the RBD, S-GCN4, and SΔC-GCN4 demonstrating that samples are pure and do not form aggregates. Only UV A280 is shown for the RBD because it is too small for light scattering to be detected with the miniDAWN detector.
. CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 28, 2020. . https://doi.org/10.1101/2020.08.28.272518 doi: bioRxiv preprint . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 28, 2020. . https://doi.org/10.1101/2020.08.28.272518 doi: bioRxiv preprint Figure S4. ELISA confirms that the ACE2 binding site and mAb epitopes are displayed on spike ferritin similarly to their display in the RBD and spike trimers. (A) BLI shows that antigens do not bind non-specifically to an off-target Ebola-specific monoclonal antibody, ADI-15731, confirming the specificity of observed binding shown in Figure 3D. (B) For ELISA, antigens were hydrophobically plated at 2 µg/mL and binding of human ACE2 and a set of SARS-CoV-2 antibodies was assessed. ELISA reveals that ACE2 and mAbs bind all antigens in a similar manner. Dilution series of hACE2 and mAbs starting at 10 µg/mL were bound to coated antigens. Binding was quantified using an anti-human-Fc HRP secondary. Each binding curve represents the average binding from 4 replicates and error bars are the standard deviation.
. CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 28, 2020. . https://doi.org/10.1101/2020.08.28.272518 doi: bioRxiv preprint Figure S5. Validation of a SARS-CoV-2 neutralization assay using a spike-pseudotyped lentivirus. The spike pseudotyped lentivirus assay was validated using two published SARS-CoV-2 neutralizing mAbs (CB6 (55) and COVA2-15 (5)) and one SARS-CoV-2 reactive mAb known to be non-neutralizing (CR3022) (52, 53). CB6 and COVA2-15 dilution curves were fit with a three-parameter non-linear regression to obtain IC50 values (Methods). Neutralization assays were performed in technical duplicate or triplicate in 4 independent experiments and one representative curve is shown. Mean IC50 values from replicates are shown in the table with standard deviation.
. CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 28, 2020. . https://doi.org/10.1101/2020.08.28.272518 doi: bioRxiv preprint Figure S6. Sera from mice immunized with SARS-CoV-2 block ACE2 binding to RBD, as indicated by ELISA. (A) ACE2 blocking activity of sera from mice immunized with a single dose of antigen was determined using an RBD-based ELISA in which RBD-coated plates were incubated with serum dilutions and then ACE2 binding was assayed. ACE2 blocking at a 1:50 and 1:500 serum dilution is shown for each group, and indicates that following a single dose, minimal ACE2 blocking activity is seen in the serum even at a high concentration. No groups show detectable ACE2 blocking activity in the serum diluted at 1:500. Each point represents the average % ACE2 blocking for a single animal assayed in duplicate; each bar represents the mean % ACE2 blocking from the group (n = 10 mice per group); error bars represent standard deviation. (B) ACE2 blocking activity was assessed after two doses of antigen and indicates a notable increase in serum antibodies capable of blocking ACE2 binding to RBD. Nearly all ACE2 binding was blocked with a 1:50 serum dilution from all groups, and all groups had detectable blocking at 1:500. Groups and error are as defined in (A).
. CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 28, 2020. . https://doi.org/10.1101/2020.08.28.272518 doi: bioRxiv preprint Table S1. Statistical analysis of spike and RBD ELISA titers from day 21 and day 28 immunization timepoints. Calculated EC50 values for each animal for RBD and spike at each time point were compiled by group and assessed using a Kruskal-Wallis ANOVA followed by Dunn's multiple comparisons test. Pairwise comparisons are shown.
. CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 28, 2020. . https://doi.org/10.1101/2020.08.28.272518 doi: bioRxiv preprint Table S2. Statistical analysis of spike and RBD neuralization titers from day 21 and day 28 immunization timepoints Calculated neutralization IC50 values for each animal at each time point were compiled by group and assessed using a Kruskal-Wallis ANOVA followed by Dunn's multiple comparisons test. Pairwise comparisons are shown.
. CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 28, 2020. . https://doi.org/10.1101/2020.08.28.272518 doi: bioRxiv preprint