Mosaic RBD nanoparticles protect against multiple sarbecovirus challenges in animal models

Abstract

To combat future SARS-CoV-2 variants and spillovers of SARS-like betacoronaviruses (sarbecoviruses) threatening global health, we designed mosaic nanoparticles presenting randomly-arranged sarbecovirus spike receptor-binding domains (RBDs) to elicit antibodies against conserved/relatively-occluded, rather than variable/immunodominant/exposed, epitopes. We compared immune responses elicited by mosaic-8 (SARS-CoV-2 and seven animal sarbecoviruses) and homotypic (only SARS-CoV-2) RBD-nanoparticles in mice and macaques, observing stronger responses elicited by mosaic-8 to mismatched (not on nanoparticles) strains including SARS-CoV and animal sarbecoviruses. Mosaic-8 immunization showed equivalent neutralization of SARS-CoV-2 variants including Omicron and protected from SARS-CoV-2 and SARS-CoV challenges, whereas homotypic SARS-CoV-2 immunization protected only from SARS-CoV-2 challenge. Epitope mapping demonstrated increased targeting of conserved epitopes after mosaic-8 immunization. Together, these results suggest mosaic-8 RBD-nanoparticles could protect against SARS-CoV-2 variants and future sarbecovirus spillovers.

Figure 1.

Mosaic nanoparticles may preferentially induce cross-reactive antibodies through avidity effects. (A) Left: Structure of SARS-2 S trimer (PDB 6VYB) showing one “up” RBD (dashed circle). Right: Sequence conservation of the 16 sarbecovirus RBDs in panel D calculated by the ConSurf Database (79) shown on two views of an RBD surface (PDB 7BZ5). The ACE2 binding footprint (PDB 6M0J) is outlined by a yellow dotted line. Locations of residues that are substituted in SARS-2 variants of concern (VOCs) and variants of interest (VOIs) as of March 2022 (https://viralzone.expasy.org/9556) are indicated as black dots. Class 1, 2, 3, 4, and 1/4 epitopes are outlined in different colored dotted lines using information from structures of representative monoclonal antibodies bound to RBD or S trimer (C102: PDB 7K8M; C002: PDB 7K8T, S309: PDB 7JX3; CR3022: PDB 7LOP; C022: PDB 7RKU). The N-linked glycan attached to RBD residue 343 is indicated by teal spheres, and the potential N-linked glycosylation site at position 370 in RBDs derived from sarbecoviruses other than SARS-2 is indicated by a teal circle. (B) Schematic showing hypothesis for how mosaic RBD-nanoparticles could induce production of cross-reactive antibodies. Left: Clustered membrane-bound B cell receptors bind with avidity to a strain-specific epitope (dark pink triangle) on dark pink antigens attached to a homotypic particle. Middle: B-cell receptors cannot bind with avidity to strain-specific epitope (triangle) on dark pink antigen attached to a mosaic particle. Right: B-cell receptors can bind with avidity to common epitope (green circle) presented on different antigens attached to a mosaic particle, but not to strain-specific epitopes (triangles). (C) Sarbecoviruses from which the RBDs in mosaic-8b RBD-mi3 were derived (matched) and sarbecoviruses from which RBDs were not included in mosaic-8b (mismatched). Clades are defined as in (13). The Wuhan-Hu-1 SARS-2 RBD was used in mosaic-8gm instead of the SARS-2 Beta RBD. (D) Phylogenetic tree of selected sarbecoviruses calculated using PhyML 3.0 (80) based on amino acid sequences of RBDs aligned using Clustal Omega (81). Viruses with RBDs included in mosaic-8b are highlighted in gray rectangles.

Figure 2.

Mosaic-8b and homotypic SARS-2 Beta RBD-mi3 immunizations induced binding and neutralizing antibodies in K18 mice. (A) Left: Immunization schedule. K18-hACE2 mice were immunized with either mosaic-8b, mosaic-8gm, homotypic SARS-2 Beta, or unconjugated SpyCatcher003-mi3 nanoparticles. Right: Structural models of mosaic-8 and homotypic RBD-mi3 nanoparticles constructed using PDB 7SC1 (RBD), PDB 4MLI (SpyCatcher), and PDB 7B3Y (mi3). (B-I) ELISA and neutralization data from Day 42 (14 days post-Boost) for antisera from individual mice (open circles) presented as the mean (bars) and standard deviation (horizontal lines). ELISA results are shown as midpoint titers (EC₅₀ values); neutralization results are shown as half-maximal inhibitory dilutions (ID₅₀ values). Dashed horizontal lines correspond to the background values representing the limit of detection. Significant differences between cohorts linked by horizontal lines are indicated by asterisks: p<0.05 = *, p<0.01 = **, p<0.001 = ***, p<0.0001 = ****. Rectangles below ELISA and neutralization data indicate mismatched strains (pink; the RBD from that strain was not present on the nanoparticle) or matched strains (gray; the RBD was present on the nanoparticle).

Figure 3.

Mosaic-8b immunization protected against SARS-2 and SARS-1 challenges in K18-hACE2 mice, whereas homotypic SARS-2 immunization protected only against SARS-2. Mice were immunized and boosted with the indicated mi3 nanoparticles represented by different colors. (A) Weight changes after SARS-2 Beta or SARS-1 challenge. Mean weight in each vaccinated cohort indicated with a thick colored line. Weights of individual mice are indicated by colored dashed lines. (B) Survival after SARS-2 Beta or SARS-1 challenge. (C) Left: SARS-2 Beta infectious titers after challenge in lung tissue and oropharyngeal swabs. Right: Genomic and subgenomic SARS-2 Beta RNA copes determined by RT-PCR. (D) Left: SARS-1 infectious titers after challenge in lung tissue and oropharyngeal swabs. Right: Genomic and subgenomic SARS-1 RNA copies determined by RT-PCR. Significant differences between cohorts linked by horizontal lines are indicated by asterisks: p<0.05 = *, p<0.01 = **, p<0.001 = ***, p<0.0001 = ****.

Figure 4.

Mosaic-8b RBD-mi3 immunization induced binding and neutralizing antibodies in NHPs. Mismatched viruses are indicated by pink rectangular boxes. (A) Left: Immunization schedule. NHPs were primed and boosted with mosaic-8b RBD-mi3 in alum and boosted again with mosaic-8b RBD-mi3 in MF59. 8 immunized NHPs and 8 unimmunized NHPs were then challenged with either SARS-2 Delta (4 immunized and 4 unimmunized) or with SARS-1 (4 immunized and 4 unimmunized). Right: Structural model of mosaic-8b RBD-mi3 nanoparticles as shown in Fig. 2A. (B-D) Viruses for assays indicated as different colors; all were mismatched with respect to mosaic-8b RBD-mi3 except for SARS-2 Beta. ELISA and neutralization data for antisera from individual NHPs (open circles) presented as the mean (bars) and standard deviation (horizontal lines). ELISA results are shown as midpoint titers (EC₅₀ values); neutralization results are shown as half-maximal inhibitory dilutions (ID₅₀ values). Dashed horizontal lines correspond to the background values representing the limit of detection.

Figure 5.

Mosaic-8b immunization protected NHPs against SARS-2 Delta and SARS-1 challenges. NHPs were immunized with mosaic-8b RBD-mi3 or not immunized (control) before challenge. (A) SARS-2 Delta infectious titers after challenge in BAL (left) and nasal swabs (right). Individual animals are denoted with different colors. (B) SARS-1 infectious titers after challenge in BAL (left) and nasal swabs (right). Individual NHPs in the unvaccinated control group are denoted with different colors to show that all four animals exhibited signs of detectable SARS-1 infectious virus in BAL and/or nasal swabs. Significant differences between cohorts linked by horizontal lines are indicated by asterisks: p<0.05 = *, p<0.01 = **, p<0.001 = ***, p<0.0001 = ****.

Figure 6.

Antibodies elicited by mosaic-8b immunization map to conserved RBD epitopes, as compared to antibodies elicited by homotypic SARS-2 Beta immunization. (A) Deep mutational scanning was used to identify mutations that reduced binding of sera from BALB/c mice immunized with mosaic-8b RBD-mi3 (top) or homotypic SARS-2 Beta RBD-mi3 (bottom) to the SARS-2 Beta RBD. The y-axis shows the site-total antibody escape (sum of the antibody escape of all mutations at a site), with larger numbers indicating more antibody escape. Each light gray line represents one antiserum, and the heavy black lines indicate the average across the n=6 sera per group. RBD sites 340–408 and 462–468, which include the more conserved class 3/4 epitopes, are indicated with solid gray lines, and sites 472–503, which include sites from the more variable class 1/2 epitopes, are indicated with dashed lines. Note that the “conserved” and “variable” epitopes presented here were generalized for simple visualization and are not identical to more specific epitope-class definitions (26, 59). The highly variable RBD class 2 site 484 that is immunodominant among humans infected with SARS-2 (44, 59) and the subdominant class 4 sites 383–386 are labeled. (B) The average site-total antibody escape for mice immunized with mosaic-8b RBD-mi3 (top) or homotypic SARS-2 RBD-mi3 (bottom) mapped to the surface of the SARS-2 Beta RBD (PDB 7LYQ), with white indicating no escape, and red indicating sites with the most escape. Key sites are labeled, all of which are class 3/4 sites, except for the class 2 484 site. Interactive logo plots and structure-based visualizations of the antibody-escape maps are at https://jbloomlab.github.io/SARS-CoV-2-RBD_Beta_mosaic_np_vaccine/. Individual antibody-escape maps are in fig. S6; raw data are in Data S2 and at https://github.com/jbloomlab/SARS-CoV-2-RBD_Beta_mosaic_np_vaccine/blob/main/results/supp_data/all_raw_data.csv. (C) Top: Residues in a “down” RBD that contact other regions of spike shown in blue on an RBD surface (PDB 7BZ5). Interacting residues were identified using the PDBePISA software server (https://www.ebi.ac.uk/pdbe/prot_int/pistart.html) and the RBD from chain A of the spike trimer structure in PDB 7M6E. Middle: variable to conserved sarbecovirus sequence gradient (dark pink = variable; green = conserved) shown on RBD surface as in Fig. 1A. Bottom: Structure of SARS-2 S trimer (PDB 6VYB) showing “down” RBD (boxed) colored with the variable to conserved sarbecovirus sequence gradient.