SARS-CoV-2 ferritin nanoparticle vaccines elicit broad SARS coronavirus immunogenicity

Abstract

The need for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) next-generation vaccines has been highlighted by the rise of variants of concern (VoCs) and the long-term threat of emerging coronaviruses. Here, we design and characterize four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of the prefusion SARS-CoV-2 spike (S), S1, and receptor-binding domain (RBD). These immunogens induce robust S binding, ACE2 inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2. A spike-ferritin nanoparticle (SpFN) vaccine elicits neutralizing titers (ID₅₀ > 10,000) following a single immunization, whereas RBD-ferritin nanoparticle (RFN) immunogens elicit similar responses after two immunizations and also show durable and potent neutralization against circulating VoCs. Passive transfer of immunoglobulin G (IgG) purified from SpFN- or RFN-immunized mice protects K18-hACE2 transgenic mice from a lethal SARS-CoV-2 challenge. Furthermore, S-domain nanoparticle immunization elicits ACE2-blocking activity and ID₅₀ neutralizing antibody titers >2,000 against SARS-CoV-1, highlighting the broad response elicited by these immunogens.

Keywords: ALFQ; B.1.1.7; B.1.351; COVID-19; P.1; SARS-CoV-1; SARS-CoV-2; betacoronaviruses; ferritin nanoparticle; neutralizing antibodies; receptor-binding domain; spike; variants of concern.

Graphical abstract

Figure 1

Structure-based design of SARS-CoV-2 S-based ferritin nanoparticle immunogens (A) Full-length SARS-CoV-2 S schematic and 3D structure. S hinges identified by molecular dynamics simulations and electron cryotomography are labeled on the 3D model (Turoňová et al., 2020). The structured trimeric ectodomain is colored according to the schematic with the N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 polypeptide and the C-terminal coiled coil N-terminal to hinge 1 colored blue, green, and purple, respectively. Remaining portions of the S1 and S2 polypeptides are colored in red and cyan with regions membrane proximal from hinge 2 colored in white. The transmembrane domain of all chains is depicted in yellow. To design a spike-ferritin molecule, the C-terminal heptad repeat (residues 1140–1161) between hinges 1 and 2 was aligned to an ideal heptad-repeat sequence. Residues in the native spike sequence that break this pattern are highlighted in red. These residues are also labeled and highlighted in red on the 3D structure. Two engineered designs (1B-05 and 1B-06) are shown, with the spike end residue used to link to ferritin, and heptad-repeat mutations colored green. (B) Schematic and 3D model of a spike-ferritin nanoparticle (SpFN). Differences between the native spike sequence and the engineered nanoparticle are indicated on the schematic. A 3D model of SpFN displaying eight trimeric spikes was created using PDB: 6VXX and 3EGM with the ferritin molecule shown in alternating gray and white. The nanoparticle is depicted along the four-fold and three-fold symmetry axes of the ferritin. (C) RBD-ferritin nanoparticle design and optimization. The RBD of SARS-CoV-2 (PDB: 6MOJ) is shown in surface representation, with the ACE2-binding site outlined in dashed lines. Three hydrophobic regions of the RBD that were mutated for nanoparticle immunogen design are shown in light green surface, with residues in stick representation. The ACE2-binding site contains two of these regions, whereas a third hydrophobic patch near the C terminus of the RBD is typically buried by S2 and part of S1 in the context of the trimer molecule. (D) Schematic and 3D model of an RBD-ferritin nanoparticle. A modeled 24-mer nanoparticle displaying the RBD domain is depicted at the three-fold symmetry axis of ferritin and colored green. Truncation points, linkers, and alterations made to the RBD sequence are indicated on the schematic. (E) Schematic and 3D model of an RBD-NTD-ferritin nanoparticle. A modeled nanoparticle displaying RBD and NTD epitopes is depicted and colored according to the schematic. Truncation points, linkers, and alterations made to the native spike sequence are indicated on the schematic. (F) S1-ferritin immunogen design. The SARS-CoV-2 S1 forms a hydrophobic collar around the N-terminal β sheet of S2 (residues 689–676). S1-ferritin immunogen design required inclusion of this short stretch of S2 (colored cyan) attached by a linker. Terminal residues of the structured portions of S1 and S2 are labeled. (G) Schematic and 3D model of an S1-ferritin nanoparticle. A modeled nanoparticle displaying RBD and NTD domains is depicted and colored according to the S1-ferritin schematic with truncation points and domain linkers indicated. See also Figure S1 and Table S1.

Figure 2

Biophysical characterization of SARS-CoV-2 S-based ferritin nanoparticle vaccine candidates (A–D) SDS-PAGE of (A) spike-ferritin nanoparticles, (B) RBD-ferritin nanoparticles, (C) RBD-NTD-ferritin nanoparticles, and (D) S1-ferritin nanoparticles. Molecular weight standards are indicated in kDa. (E) Size-exclusion chromatography on a Superdex S200 10/300 column of representative SARS-CoV-2 S-based ferritin nanoparticles. (F) Negative-stain electron microscopy 2D class averages of purified nanoparticles. The scale bars represent 50 nm. See also Figures S2 and S3.

Figure 3

Antigenic characterization of select SARS-CoV-2 S-based ferritin nanoparticle vaccine candidates Binding response of SARS-CoV-2 neutralizing antibodies to each of the lead candidates from the four design categories measured by biolayer interferometry with two-fold serial dilution of each antibody starting at 30 μg/mL. (A) Spike-ferritin nanoparticle SpFN_1B-06-PL. (B) RBD-ferritin nanoparticle RFN_131. (C) RBD-NTD-ferritin nanoparticle pCoV146. (D) S1-ferritin nanoparticle pCoV111. See also Figure S3.

Figure 4

Negative-stain electron microscopy 3D reconstructions of SARS-CoV-2 S-based ferritin nanoparticles Modifications made to the native sequence and linkers used for each construct are shown in schematic diagrams. The size of each particle is indicated in nanometers. (A) Negative-stain 3D reconstructions with applied octahedral symmetry are shown with an asymmetric unit of non-ferritin density colored. Spike trimer density is colored in red, and a model of a SARS-CoV-2 S-2P trimer based on PDB: 6VXX is shown docked into the negative-stain map and colored according to the schematic diagram. (B) Two non-ferritin densities per asymmetric unit were observed for RFN_131 and are highlighted in green. These densities putatively correspond to the RBD but lack low-resolution distinguishing features due to the small, globular shape of these domains. The presence of two densities is likely due to flexibility in the linker and heterogeneity in the RBD pose. (C) Two layers of densities were distinguishable for pCoV146, with the putative NTD density of an asymmetric unit colored blue, proximal to the ferritin, and two smaller, more flexible densities corresponding to the RBD distal to the ferritin and colored green. (D) An asymmetric unit of non-ferritin density for pCoV111 is colored in orange and a monomer of S1 in the closed trimer state from PDB: 6VXX is shown docked into the density with domains colored as in the schematicdiagram. See also Figure S2 and Table S2.

Figure 5

SARS-CoV-2 S-domain nanoparticle vaccine candidates elicit robust binding and neutralizing antibody responses in C57BL/6 mice Data relating to each category of immunogen are colored as follows: SpFN_1B-06-PL, blue; RFN_131, green; pCoV146, black; and pCoV111, orange. n = 10/group. ALFQ was the adjuvant used for these animal groups. (A and B) ELISA binding of mouse sera to SARS-CoV-2 RBD or S-2P. Study week is indicated at the base of the graph. Geometric mean value is indicated by a horizontal line. Statistical comparison at each time point was carried out using a Kruskal-Wallis test followed by a Dunn’s post-test. (C) SARS-CoV-2 pseudovirus neutralization ID₅₀ values. Geometric mean titer values are indicated by a horizontal line. Statistical comparison at each given time point was carried out using a Kruskal-Wallis test followed by a Dunn’s post-test. (D) ELISA analysis of antibody isotype usage following immunization with SpFN + ALFQ (solid shapes) or SpFN + Alhydrogel (open shapes). Sera collected at study week 2, 5, and 8 from immunized mice were added in quadruplicate serial dilutions to ELISA plates coated with S-2P protein. Duplicated wells were probed with anti-mouse-IgG1-HRP. Additional duplicates were probed with either anti-mouse-IgG2c-HRP or anti-mouse IgG2a-HRP for C57BL/6 and BALB/c mice, respectively. Data were interpolated to obtain the dilution factor at OD₄₅₀ of 1 and plotted as ratios of IgG2/IgG1. A horizontal dotted line denotes a balanced 1:1 IgG2/IgG1 ratio. Isotype ratio values were compared between the two adjuvant groups at each timepoint for each mouse type using a Mann-Whitney unpaired two-tailed non-parametric test. (E) Binding and pseudovirus neutralization of sera from mice immunized with 0.08 μg SpFN + ALFQ. (F) Authentic SARS-CoV-2 virus neutralization ID₅₀ values are shown for mice immunized with 10 μg (blue) or 0.08 μg (light blue) SpFN + ALFQ. Geometric mean titer is indicated by a horizontal line. Comparisons between dose groups at each time point were carried out using a Mann-Whitney unpaired two-tailed non-parametric test. In (B) and (C), all groups at a given study time point were compared to each other. Only groups with significant differences are indicated by a bar; all other groups did not show statistically significant differences. ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05. See also Figures S4–S6 and Tables S3 and S4.

Figure 6

SARS-CoV-2 S-domain nanoparticle vaccine candidates elicit robust antibody binding responses and neutralizing activity against SARS-CoV-2 VoCs and SARS-CoV-1 (A) Pseudovirus neutralization (ID₅₀ values) of study week 10 mouse sera from immunized C57BL/6 and BALB/c mice to SARS-CoV-2 Wuhan-1, B.1.1.7, and B.1.351 pseudotyped viruses. Immunogens are indicated at the base of each graph. Geometric mean titer values are indicated by a horizontal line; n = 5; statistical significance for each immunogen was assessed using a Kruskal-Wallis test followed by a Dunn’s post-test. (B) Biolayer interferometry binding of study week 10 mouse sera from immunized C57BL/6 and BALB/c mice to SARS-CoV-1 RBD. Immunogens are indicated at the base of each graph. Mean values are indicated by a horizontal line; n = 10; statistical significance was assessed using a Kruskal-Wallis test followed by a Dunn’s post-test. (C) Pseudovirus neutralization (ID₅₀ values) of study week 10 mouse sera from immunized C57BL/6 and BALB/c mice to SARS-CoV-1 Urbani strain pseudotyped viruses. Data related to SpFN_1B-06-PL and RFN_131 are colored blue and green, respectively. Statistical comparisons between SpFN and RFN responses at each time point were carried out using a Mann-Whitney unpaired two-tailed non-parametric test. Immunogens are indicated at the base of each graph. Geometric mean values are indicated by a horizontal line; n = 10; ^∗∗∗∗p < 0.0001, ^∗∗p < 0.01, ^∗p < 0.05. See also Figures S4 and S5.

Figure 7

SpFN- and RFN-protective immunity in K18-hACE2 transgenic mice (A) IgG was purified from SpFN- or RFN-vaccinated mouse sera and passively transferred at specific IgG amounts ranging from 4 to 470 μg/mouse in a final volume of 200 μL. Control naive mouse IgG was formulated at 2 mg/mL (n = 10/group, 5 female, 5 male). (B) Mouse challenge study schematic. K18-hACE2 mice (n = 10/group, 5 female, 5 male) received control IgG (black), PBS (gray), and purified IgG, 1 day prior to challenge with 1.25 × 10⁴ PFU of SARS-CoV-2. (C) SARS-CoV-2 pseudovirus neutralization ID₅₀ titers of mouse sera at study day 0. Pseudovirus IC₅₀ geometric mean titer for each group is shown on the x axis. The horizontal dotted line indicates the lower limit of detection of the assay. (D and E) Percentage of initial weight (D) and survival (E) of K18-hACE2 mice for the 8 study groups. Groups are indicated based on original animal vaccination group and the pseudovirus ID₅₀ neutralization values. The legend is shown in (D). Statistical comparisons were carried out using a Mantel-Cox test followed by Bonferroni correction. See also Figure S7.