Elicitation of potent neutralizing antibody responses by designed protein nanoparticle vaccines for SARS-CoV-2

Abstract

A safe, effective, and scalable vaccine is urgently needed to halt the ongoing SARS-CoV-2 pandemic. Here, we describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 copies of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) in a highly immunogenic array and induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes on the RBD, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the protein components and assembled nanoparticles, especially compared to the SARS-CoV-2 prefusion-stabilized S trimer, suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms for inducing potent neutralizing antibody responses and have launched cGMP manufacturing efforts to advance the lead RBD nanoparticle vaccine into the clinic.

Figure 1.. Design, In Vitro Assembly, and Characterization of SARS-CoV-2 RBD Nanoparticle Immunogens

(A) Molecular surface representation of the SARS-CoV-2 S-2P trimer in the prefusion conformation (PDB 6VYB). Each protomer is colored distinctly, and N-linked glycans are rendered dark blue (the glycan at position N343 was modeled based on PDB 6WPS and the receptor-binding motif (RBM) was modeled from PDB 6M0J). The single open RBD is boxed. (B) Molecular surface representation of the SARS-CoV-2 S RBD, including the N-linked glycans at positions 331 and 343. The ACE2 receptor-binding site or RBM is indicated with a black outline. (C) Structural models of the trimeric RBD-I53–50A (RBD in light blue and I53–50A in light gray) and pentameric I53–50B (orange) components. Upon mixing in vitro, 20 trimeric and 12 pentameric components assemble to form nanoparticle immunogens with icosahedral symmetry. Each nanoparticle displays 60 copies of the RBD. (D) Structural model of the RBD-12GS-I53–50 nanoparticle immunogen. Although a single orientation of the displayed RBD antigen and 12-residue linker are shown for simplicity, these regions are expected to be flexible relative to the I53–50 nanoparticle scaffold. (E) Dynamic light scattering (DLS) of the RBD-8GS-, RBD-12GS-, and RBD-16GS-I53–50 nanoparticles compared to unmodified I53–50 nanoparticles. (F) Representative electron micrographs of negatively stained RBD-8GS-, RBD-12GS-, and RBD-16GS-I53–50 nanoparticles. The samples were imaged after one freeze/thaw cycle. Scale bars, 100 nm. (G) Hydrogen/Deuterium-exchange mass spectrometry of monomeric RBD versus trimeric RBD-8GS-I53–50A component, represented here as a butterfly plot, confirms preservation of the RBD conformation, including at epitopes recognized by known neutralizing Abs. In the plot, each point along the horizontal sequence axis represents a peptide where deuterium uptake was monitored from 3 seconds to 20 hours. Error bars shown on the butterfly plot indicate standard deviations from two experimental replicates. The difference plot below demonstrates that monomeric RBD and RBD-8GS-I53–50A are virtually identical in local structural ordering across the RBD. (H) Pie charts summarizing the glycan populations present at the N-linked glycosylation sites N331 and N343 in five protein samples: monomeric RBD, S-2P trimer, and RBD-8GS-, RBD-12GS-, and RBD-16GS-I53–50A trimeric components. The majority of the complex glycans at both sites were fucosylated; minor populations of afucosylated glycans are set off by dashed white lines. Oligo, oligomannose.

Figure 2.. Antigenic Characterization of SARS-CoV-2 RBD-I53–50 Nanoparticle Immunogens

(A) Bio-layer interferometry of immobilized mACE2-Fc, CR3022 mAb, and S309 mAb binding to RBD-8GS-, RBD-12GS-, and RBD-16GS-I53–50 nanoparticles displaying the RBD antigen at 50% or 100% valency. The monomeric SARS-CoV-2 RBD was included in each experiment as a reference. (B) The binding signal at 880 s, near the end of the association phase, is plotted for each experiment in panel (A) to enable comparison of the binding signal obtained from each nanoparticle.

Figure 3.. Physical and Antigenic Stability of RBD Nanoparticle Immunogens and S-2P Trimer

(A) Chemical denaturation by guanidine hydrochloride. The ratio of intrinsic tryptophan fluorescence emission at 350/320 nm was used to monitor protein tertiary structure. Major transitions are indicated by shaded regions. Representative data from one of three independent experiments are shown. (B) Summary of SDS-PAGE and nsEM stability data over four weeks. SDS-PAGE showed no detectable degradation in any sample. nsEM revealed substantial unfolding of the S-2P trimer at 2–8°C after three days incubation, and at 22–27°C after four weeks. N/A, not assessed. (C) Summary of antigenicity data over four weeks. The antigens were analyzed for mACE2-Fc (solid lines) and CR3022 mAb (dashed lines) binding by bio-layer interferometry after storage at the various temperatures. The plotted value represents the amplitude of the signal near the end of the association phase normalized to the corresponding <−70°C sample at each time point. (D) Summary of UV/vis stability data over four weeks. The ratio of absorbance at 320/280 nm is plotted as a measure of particulate scattering. Only the S-2P trimer and the RBD-12GS-I53–50 nanoparticle showed any increase in scattering, and only at ambient temperature. (E) DLS of the RBD-12GS-I53–50 nanoparticle indicated a monodisperse species with no detectable aggregate at all temperatures and time points. The data in panels B-E is from a four-week real-time stability study that was performed once.

Figure 4.. RBD-I53–50 Nanoparticle Immunogens Elicit Potent Antibody Responses in BALB/c and Human Immune Repertoire Mice

(A-B) Post-prime (week 2) (A) and post-boost (week 5) (B) anti-S binding titers in BALB/c mice, measured by ELISA. Each symbol represents an individual animal, and the geometric mean from each group is indicated by a horizontal line. The dotted line represents the lower limit of detection of the assay. 8GS, RBD-8GS-I53–50; 12GS, RBD-12GS-I53–50; 16GS, RBD-16GS-I53–50; HCS, human convalescent sera. The inset depicts the study timeline. The immunization experiment was repeated twice and representative data are shown. (C-D) Post-prime (week 2) (C) and post-boost (week 5) (D) anti-S binding titers in Kymab Darwin mice, which are transgenic for the non-rearranged human antibody variable and constant region germline repertoire, measured by ELISA and plotted as in (A). The inset depicts the study timeline. The immunization experiment was performed once. Statistical analyses are provided in Supplementary Item 4.

Figure 5.. RBD-I53–50 Nanoparticle Immunogens Elicit Potent and Protective Neutralizing Antibody Responses

(A-B) Serum pseudovirus neutralizing titers post-prime (A) or post-boost (B) from mice immunized with monomeric RBD, S-2P trimer, or RBD-I53–50 nanoparticles. Each circle represents the reciprocal IC₅₀ of an individual animal. The geometric mean from each group is indicated by a horizontal line. Limit of detection shown as a gray dotted line. The animal experiment was performed twice, and representative data from duplicate measurements are shown. (C-D) Serum live virus neutralizing titers post-prime (C) or post-boost (D) from mice immunized as described in (A). (E-F) Serum pseudovirus neutralizing titers from Kymab Darwin mice post-prime (E) and post-boost (F), immunized as described in (A). The animal experiment was performed once, and the neutralization assays were performed at least in duplicate. (G-H) Seven weeks post-boost, eight BALB/c mice per group were challenged with SARS-CoV-2 MA. Two days post-challenge, viral titers in lung tissue (G) and nasal turbinates (H) were assessed. Limit of detection depicted as a gray dotted line. Statistical analyses are provided in Supplementary Item 4.

Figure 6.. RBD Nanoparticle Vaccines Elicit Robust B Cell Responses and Antibodies Targeting Multiple Epitopes in Mice and a Nonhuman Primate

(A-B) Number of (A) RBD⁺ B cells (B220⁺CD3⁻CD138⁻) and (B) RBD⁺ GC precursors and B cells (CD38^+/−GL7⁺) detected across each immunization group. (C-D) Frequency of (C) RBD⁺ GC precursors and B cells (CD38^+/−GL7+) and (D) IgD⁺, IgM⁺, or class-switched (IgM⁻IgD⁻; swIg⁺) RBD⁺ GC precursors and B cells. (A-D) N=6 across two experiments for each group. Statistical significance was determined by one-way ANOVA, and Tukey’s multiple comparisons tests were performed for any group with a p-value less than 0.05. Significance is indicated with stars: * p < 0.05, **** p < 0.0001. (E) Ratio post-boost (week 5) of S-2P ELISA binding titer (Figure 4D) to pseudovirus neutralization titers (Figure 5F) in Kymab Darwin mice. The ratio is the [GMT (EC₅₀) of five mice]:[the GMT (IC₅₀) of five mice] or the EC₅₀:|C₅₀ of all HCS tested. A lower value signifies a higher quality response. (F) Ratio post-boost (week 5) of S-2P ELISA binding titer (Figure 4B) to either pseudovirus (Figure 5B) or live virus (Figure 5D) neutralization titers in BALB/c mice. The ratio is the [GMT (EC₅₀) of ten mice]:[the GMT (IC₅₀) of ten mice] or the EC₅₀:|C₅₀ of all HCS tested. (G) SARS-CoV-2 RBD (gray ribbon) with monomeric ACE2 (blue surface), CR3022 Fab (green surface), and S309 Fab (red surface) bound. (H-J) Determination of vaccine-elicited Ab epitope specificity by competition BLI. A dilution series of polyclonal NHP Fabs was pre-incubated with RBD on the BLI tip. The polyclonal Fab concentration was maintained with the addition of competitor to each dilution point. The 1:3 dilution series of polyclonal Fabs is represented from dark to light, with a dark gray line representing competitor loaded to apo-RBD (no competition). Competition with (H) 200 nM ACE2, (I) 400 nM CR3022, or (J) 20 nM S309.