Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines

Abstract

Understanding vaccine-elicited protection against SARS-CoV-2 variants and other sarbecoviruses is key for guiding public health policies. We show that a clinical stage multivalent SARS-CoV-2 spike receptor-binding domain nanoparticle (RBD-NP) vaccine protects mice from SARS-CoV-2 challenge after a single immunization, indicating a potential dose-sparing strategy. We benchmarked serum neutralizing activity elicited by RBD-NPs in non-human primates against a lead prefusion-stabilized SARS-CoV-2 spike (HexaPro) using a panel of circulating mutants. Polyclonal antibodies elicited by both vaccines are similarly resilient to many RBD residue substitutions tested, although mutations at and surrounding position 484 have negative consequences for neutralization. Mosaic and cocktail nanoparticle immunogens displaying multiple sarbecovirus RBDs elicit broad neutralizing activity in mice and protect mice against SARS-CoV challenge even in the absence of SARS-CoV RBD in the vaccine. This study provides proof of principle that multivalent sarbecovirus RBD-NPs induce heterotypic protection and motivates advancing such broadly protective sarbecovirus vaccines to the clinic.

Keywords: COVID-19; SARS-CoV-2; mosaic nanoparticle; receptor-binding domain; sarbecovirus; self-assembling nanoparticle; spike glycoprotein; vaccine design.

Graphical abstract

Figure 1

A single immunization with RBD-NP protects BALB/c cByJ mice from SARS-CoV-2 MA10 challenge (A and C) S2P-binding Abs were measured 2 (A) or 5 (C) weeks post-prime; teal squares: 2 doses of 0.1 μg (n = 10); filled teal circles: 2 doses of 1 μg (n = 8); open teal circles: one dose of 1 μg (n = 8) with a limit of detection (LOD) of 1 × 10^2. (B and D) Serum neutralizing Ab titers at 2 (B) or 5 (D) weeks post-prime determined using an MLV pseudotyping system with an LOD of 3.3 × 10¹. (E) Weight loss following SARS-CoV-2 MA10 challenge up to 4 days post infection (n = 8 vaccinated; n = 6 naive mice shown as black filled circles). (F) Congestion score following SARS-CoV-2 MA10 challenge with a score of 0 indicating unchanged lung color and 4 indicating a darkened and diseased lung. (G) Viral titers in the mice lungs (expressed in plaque forming units [PFUs] per lobe) following challenge with an LOD of 9 × 10¹. Statistical significance was determined by Kruskal-Wallis test and shown only when significant. ^∗∗p < 0.01. LODs are shown as gray horizontal dotted lines. Raw data curves shown in Data S1.

Figure 2

RBD-NP vaccination elicits high titers of Abs targeting diverse RBD antigenic sites in NHPs (A) NHP study designs. (B) Effect of RBD mutations on polyclonal Ab binding measured by DMS analysis of serum obtained 8 weeks post-prime from an immunized pigtail macaque (n = 1) compared to a previously reported DMS measurement from a representative COVID-19 HCP sample (reproduced here for comparison; Greaney et al., 2021b). The line plots on the left show the summed effect of all mutations at a site in the RBD on serum or plasma binding, with RBD residues on the x axis and Ab escape on the y axis (Table S1). Due to the use of sample-specific FACS gates (Figure S1), the y axes are scaled independently. Sites in the logo plots are colored dark blue if located in the receptor-binding ridge or cyan if located in the RBD 443–450 loop. Larger values indicate more Ab escape. (C–E) Competition ELISA between 0.2-nM hACE2-Fc (LOD of 2 × 10¹) (C), 2-nM CR3022 mAb (LOD of 2 × 10°) (D), or 0.01-nM S309 mAb (LOD of 4 × 10°) (E), and RBD-NP-elicited sera in pigtail macaques (n = 2), RBD-NP-elicited sera in rhesus macaques (n = 5), or HexaPro S-elicited sera in rhesus macaques (n = 5) at various time points following vaccination, benchmarked against HCP (n = 4) (Table S2). Each plot shows the magnitude of inhibition of hACE2/mAb binding to immobilized SARS-CoV-2 S2P, expressed as reciprocal serum dilution blocking 50% of the maximum binding response. Statistical significance was determined by Kruskal-Wallis test and shown only when significant. ^∗∗p < 0.01. LODs are shown as gray horizontal dotted lines. Raw data curves shown in Data S1.

Figure S1

Effects of mutations on binding of HCP Abs to RBD and FACS gating strategy, related to Figure 2 (A) Correlation plots of site- and mutation-level escape for each of the two independent RBD mutant libraries for the Ab-escape map shown in Figure 2B. Site-level escape is the sum of the escape fractions for each mutation at a site. (B) Hierarchical FACS gating strategy used for selecting yeast cells expressing Ab-escape RBD variants. First, gates are selected to enrich for single cells (SSC-A versus FSC-A, and FSC-W versus FSC-H) that also express RBD (FITC-A versus FSC-A, cells in pink). Second, cells expressing RBD mutants with reduced polyclonal Ab binding, detected with an anti-IgA+IgG+IgM secondary Ab, were selected with a gate that captured the ∼5% of cells with the lowest Ab binding (cells in blue).

Figure S2

Evaluation of vaccine-elicited binding and neutralizing Ab titers against SARS-CoV-2 variants and sarbecoviruses, related to Figure 3 (A) Wild-type (Wuhan-Hu-1) and B.1.351 SARS-CoV-2 RBD-specific Ab binding titers of RBD-NP-elicited sera in pigtail macaques (magenta, n = 2) and rhesus macaques (blue, n = 5), HexaPro S-elicited sera in rhesus macaques (gray, n = 5), or HCP (orange, n = 6, Table S1) analyzed by ELISA with an LOD of 1x10². (B) Cladogram based on sarbecovirus RBD amino acid sequences. (C) Biolayer interferometry analysis of binding of 1 μM purified polyclonal pigtail macaque IgGs (obtained 70 days post prime) to sarbecovirus RBDs immobilized at the surface of biosensors. (D) SARS-CoV-2 S2P (left) or SARS-CoV S2P (right) Ab binding titers of RBD-NP-elicited sera in pigtail macaques (magenta) or HexaPro S-elicited sera in rhesus macaques (gray) analyzed by ELISA with an LOD of 2.5 × 10¹. (E) Competition ELISA between 0.13 nM human ACE2-Fc and RBD-NP-elicited sera in pigtail macaques (magenta) and rhesus macaques (blue), or HexaPro S-elicited sera in rhesus macaques (gray) at various time points following vaccination, benchmarked against COVID-19 HCP with an LOD of 4 × 10°, showing the magnitude of inhibition of ACE2 binding to immobilized SARS-CoV S2P expressed as reciprocal serum dilution blocking 50% of the maximum binding response. Statistical significance was determined by Kruskal-Wallis test and shown when significant. ^∗∗, p < 0.01. All data repeated twice. LODs are shown as gray horizontal dotted lines. Raw data curves shown in Data S1.

Figure 3

RBD-NP and HexaPro S elicit Abs with similar neutralization breadth toward SARS-CoV-2 variants (A) Neutralizing Ab titers against wild-type (D614G) SARS-CoV-2 S and RBD point mutants determined using RBD-NP-elicited sera in rhesus macaques (blue, n = 5) and HexaPro S-elicited sera in rhesus macaques (gray, n = 6) with an VSV pseudotyping system with an LOD of 3.3 × 10¹. Neutralization performed once. (B) Neutralizing Ab titers against HIV pseudotyped viruses harboring wild-type (D614G) SARS-CoV-2 S, B.1.1.7 S, B.1.1.7-E484K S, B.1.351 S, or P.1 S, determined using RBD-NP-elicited sera in rhesus macaques (blue), HexaPro S-elicited sera in rhesus macaques (gray), or plasma from individuals who received two doses of Pfizer mRNA vaccine (open circles) with an LOD of 3.3 × 10¹. Neutralization performed twice and a representative shown. (C) Neutralizing Ab titers against VSV pseudotyped viruses harboring wild-type (D614G) SARS-CoV-2 S, B.1.1.7 S, B.1.351 S, or P.1 S, determined using RBD-NP-elicited sera in rhesus macaques (blue) or HexaPro S-elicited sera in rhesus macaques (gray) with an LOD of 2.5 × 10¹. Neutralization performed twice and a representative shown. (D) Neutralizing Ab titers against D614G SARS-CoV-2 S, B.1.1.7, and B.1.351 S HIV pseudoviruses in pigtail macaque sera collected 28 days after a second (filled symbols, n = 2) or third (open symbols, n = 2) immunization with 88-μg RBD-NP (RBD antigen dose) with an LOD of 1 × 10². Neutralization performed twice and a representative shown. (E) Neutralizing Ab titers against HIV pseudotyped viruses harboring wild-type (D614G) SARS-CoV-2 S, Pangolin-GD S, or SARS-CoV S, determined using RBD-NP-elicited sera in in rhesus macaques (blue) or HexaPro S-elicited sera in rhesus macaques (gray) with an LOD of 1 × 10¹. Neutralization performed twice and a representative shown. (F) Neutralizing Ab titers against VSV pseudotyped viruses harboring wild-type (D614G) SARS-CoV-2 S, Pangolin-GD S, RaTG13 S, SARS-CoV S, or WIV1 S determined using SARS-CoV-2 RBD-NP-elicited sera in rhesus macaques (blue) or HexaPro S-elicited sera in rhesus macaques (gray) with an LOD of 2.5 × 10¹. Neutralization performed twice and a representative shown. Statistical significance was determined by Kruskal-Wallis test and shown in Table S3. LODs are shown as gray horizontal dotted lines. Raw data curves shown in Data S1 and GMTs in Table S4. The various pseudovirus backbones were benchmarked against NIBSC standard and are shown in Table S5.

Figure 4

In vitro assembly and accelerated stability studies of mosaic and cocktail nanoparticle immunogens (A) Schematic of in vitro assembly of mRBD-NPs and cRBD-NPs. (B–E) The physical and antigenic stability of mRBD-NP, cRBD-NP, and (SARS-CoV-2) RBD-NP samples incubated at four different temperatures was followed for four weeks. (B) The ratio of UV/vis absorbance at 320 nm/280 nm is a measure of turbidity (proxy for aggregation). (C) Hydrodynamic diameter of the nanoparticles measured using dynamic light scattering. (D) hACE2-Fc binding measured by comparing the peak amplitude of hACE2-Fc binding for each sample to a reference sample stored at < −70°C using biolayer interferometry. (E) Electron micrographs of negatively stained samples after incubation for 28 days at the indicated temperatures. Scale bar, 50 nm.

Figure S3

In vitro characterization and confirmation of co-display of sarbecovirus RBD-NP immunogens, related to Figure 4 (A) Design models of the various vaccine candidates evaluated. Scale bars, 36 nm. (B) SDS-PAGE analysis of purified nanoparticles. DTT, dithiothreitol; F/T, freeze/thaw. (C) Dynamic light scattering. (D) Electron micrographs of negatively stained samples. Scale bars, 50 nm. (E) Binding of 100 nM SEC-purified nanoparticle immunogens and the non-assembling cocktail immunogen (which was not purified with SEC) to immobilized hACE2-Fc. (F) SEC chromatogram overlay of purified RBD-NP and non-assembling cocktail. (G-H) Sandwich biolayer interferometry. The SARS-CoV-2 S-specific mAb S2H14 immobilized on protein A biosensors was used to capture various nanoparticle immunogens from 300-480 s. The captured nanoparticles were subsequently exposed to a Fab derived from the SARS-CoV S-specific mAb S230 from 600-900 s (G). hACE2-Fc immobilized on protein A biosensors was used to capture various nanoparticle immunogens from 300-480 s. The captured nanoparticles were subsequently exposed to a Fab derived from the SARS-CoV S-specific mAb S230 from 600-900 s (H).

Figure S4

Serum Ab binding titers elicited by mosaic and cocktail RBD-NPs, related to Figure 5 (A) Ab binding titers to SARS-CoV-2 S2P at five weeks post prime analyzed by ELISA with an LOD of 1x10². (B–E) Titers of SARS-CoV-2 S-specific Abs competing with ACE2-Fc with an LOD of 5x10¹ (B), CR3022 with an LOD of 5x10¹ (C), S309 with an LOD of 1x10¹ (D), and S2X259 with an LOD of 1x10¹ (E) in immunized mouse sera analyzed by competition ELISA. (F) Ab binding titers to SARS-CoV S2P at week 5 analyzed by ELISA. (G–H) Ab binding titers to the WIV1 (G), and RaTG13 (H) RBDs analyzed by ELISA with an LOD of 1x10². Statistical significance was determined by Kruskal Wallis test and shown when significant. ^∗∗p < 0.01. LODs are shown as gray horizontal dotted lines. Raw data curves shown in Data S1.

Figure 5

Mosaic and cocktail RBD-NP vaccines elicit neutralizing Abs against multiple sarbecoviruses (A) Neutralizing Ab titers in mice (n = 8) against wild-type (D614G) SARS-CoV-2 S MLV pseudovirus five weeks post-prime elicited by monovalent, mosaic, and cocktail RBD-NPs with an LOD of 1 × 10¹. (B) Neutralizing Ab titers in mice against SARS-CoV S MLV pseudovirus five weeks post-prime elicited by monovalent, mosaic, and cocktail RBD-NPs with an LOD of 1 × 10¹. (C) Neutralizing Ab titers in mice against SHC014 VSV pseudovirus five weeks post-prime elicited by monovalent, mosaic, and cocktail RBD-NPs with an LOD of 1.7 × 10¹. Raw data curves shown in Data S1. Statistical significance was determined by Kruskal-Wallis test and shown only when significant. ∗∗p<0.01. LOD is shown as a gray horizontal dotted line in (C).

Figure 6

Mosaic and cocktail RBD-NP vaccines protect against heterotypic SARS-CoV-MA15 challenge in 15-week-old BALB/c cByJ mice (A) Weight loss following SARS-CoV MA15 challenge up to 4 days post infection (n = 8). Unvaccinated animals are shown as black circles. (B) Congestion score following SARS-CoV MA15 infection with a score of 0 indicating unchanged lung color and 4 indicating a darkened and diseased lung (n = 8). (C) Viral titers in mice lungs (expressed in PFUs per lobe) following challenge (n = 8) with an LOD of 9 × 10¹. LOD is shown as a gray horizontal dotted line. Statistical significance was determined by Kruskal-Wallis and shown when significant. ^∗∗p < 0.01. (D) Mean eosinophils per high power field (HPF) per sample run over 10 HPF per lung stained with congo red. Significance was determined using one-way ANOVA and shown where significant. (E–I) Histological analysis of stained lung sections for mRBD-NP (E), mRBD-NP-DO (F), cRBD-NP (G), cRBD-NP-DO (H), and unvaccinated mice (I). Arrowheads indicate eosinophils. Scale bar, 20 μm.

Figure S5

Monovalent, mosaic, and cocktail RBD-NPs protect against heterotypic SARS-CoV-MA15 challenge in 15-week-old BALB/c cByJ mice, related to Figure 6 (A) Normalized active inflammation following SARS-CoV MA 15 challenge shown in Figure 6 with venulitis, endarteritis, and interstitial pneumonitis shown as stacked bar graphs in dark gray, light gray, and black respectively. (B) Weight loss following SARS-CoV MA15 challenge (N = 6). Unvaccinated animals are shown as black circles. (C) Congestion score following SARS-CoV MA15 challenge with a score of 0 indicating unchanged lung color and 4 indicating a darkened and diseased lung (N = 6). (D) Viral titers in mice lungs (expressed in plaque forming units per lobe) following challenge (N = 8) with an LOD of 9x10¹. Statistical significance was determined by Kruskal Wallis test and shown when significant and ^∗∗p < 0.01. LOD is shown as a gray horizontal dotted line.