Protective prototype-Beta and Delta-Omicron chimeric RBD-dimer vaccines against SARS-CoV-2

Abstract

Breakthrough infections by SARS-CoV-2 variants become the global challenge for pandemic control. Previously, we developed the protein subunit vaccine ZF2001 based on the dimeric receptor-binding domain (RBD) of prototype SARS-CoV-2. Here, we developed a chimeric RBD-dimer vaccine approach to adapt SARS-CoV-2 variants. A prototype-Beta chimeric RBD-dimer was first designed to adapt the resistant Beta variant. Compared with its homotypic forms, the chimeric vaccine elicited broader sera neutralization of variants and conferred better protection in mice. The protection of the chimeric vaccine was further verified in macaques. This approach was generalized to develop Delta-Omicron chimeric RBD-dimer to adapt the currently prevalent variants. Again, the chimeric vaccine elicited broader sera neutralization of SARS-CoV-2 variants and conferred better protection against challenge by either Delta or Omicron SARS-CoV-2 in mice. The chimeric approach is applicable for rapid updating of immunogens, and our data supported the use of variant-adapted multivalent vaccine against circulating and emerging variants.

Keywords: COVID19 vaccine; Delta variant; Omicron variant; RBD; SARS-CoV-2; VOC; immunogen structure; receptor-binding domain; vaccine protection; variant of concern.

Graphical abstract

Figure S1

Schematic representation of SARS-CoV-2 spike proteins used in this study and analytical gel filtration profiles of RBD-dimer proteins, related to Figures 1, 2, and 5 (A) The prototype sequence is from Wuhan-1 reference strain. The mutation sites of the variants were indicated. SP, signal peptide; NTD, N-terminal domain; RBD, receptor-binding domain; TM, transmembrane domain; CTD, C-terminal domain. (B) Prototype RBD-dimer protein. (C) Beta RBD-dimer protein. (D) Prototype-Beta chimeric RBD-dimer protein. The gel filtration was carried out in HiLoad 16/600 Superdex 200 pg (A–C). (E) Delta-Omicron chimeric RBD-dimer protein. The gel filtration was carried out in Superdex 200 Increase 10/300 GL. The 280-nm absorbance curves are shown. SDS-PAGE migration was carried out in non-reduced and reduced conditions (B–E).

Figure 1

Antigenic and structural characterization of homotypic and chimeric RBD-dimers (A) Schematic diagram of prototype RBD-dimer, Beta RBD-dimer, prototype-Beta chimeric RBD-dimer, and Delta-Omicron chimeric RBD-dimer. Two SARS-CoV-2 RBDs were dimerized as tandem repeat (SP, signal peptide). (B) Footprint of hACE2 and five classes of antibodies on SARS-CoV-2 RBD. Prototype RBD is shown as gray surface (PDB: 6LZG). Residues that mutated in SARS-CoV-2 Beta and Delta variants are colored in red and blue, respectively. Footprints of hACE2 (PDB: 6LZG), CB6 (PDB: 7C01), CV07-270 (PDB: 6XKP), C110 (PDB: 7K8V), S309 (PDB: 6WPT), and CR3022 (PDB: 6W41) are highlighted in pink, blue, yellow, orange, cyan, and green, respectively. (C) Footprint of hACE2 and five classes of antibodies on SARS-CoV-2 RBD. Prototype RBD is shown as gray surface. Residues that mutated in SARS-CoV-2 Omicron variants are colored in red. (D) Binding affinities of antigens bound to hACE2 and representative mAbs targeting five major sites. Red indicates no binding (N/B). The ones with affinity reductions more than 100-fold are colored in yellow. (E) Density map of prototype RBD-dimer bound to two CB6 Fabs, with the atomic models of the SARS-CoV-2 RBD/CB6 complex (PDB:7C01) were fitted and rebuilt. (F) Density map of prototype-Beta chimeric RBD-dimer bound to one CB6 Fab, with the atomic models of SARS-CoV-2 RBD (PDB: 6LZG) and RBD/CB6 complex (PDB: 7C01) were fitted and rebuilt, clearly showing that Beta variant RBD does not bind to CB6. (G) Density map of Delta-Omicron chimeric RBD-dimer bound to one CB6 Fab, with the atomic models of Delta RBD (PDB: 7V8B), Omicron RBD (PDB: 7WBL) and CB6 (PDB: 7C01) were fitted and rebuilt, clearly showing that Omicron variant RBD does not bind to CB6.

Figure S2

BIAcore diagram of RBD-monomer and RBD-dimer proteins bound to hACE2 and mAbs, related to Figure 1 (A) Prototype RBD, Beta RBD, prototype RBD-dimer, Beta RBD-dimer, or prototype-Beta chimeric RBD-dimer proteins bound to hACE2 and mAbs. (B) Prototype RBD, Delta RBD, Omicron RBD, or Delta-Omicron chimeric RBD-dimer proteins bound to hACE2 and mAbs. The antigen proteins were immobilized on the chip and were tested for binding with gradient concentrations of hACE or mAb Fabs as indicated using multi-cycle mode by BIAcore 8000. The binding profiles are shown with time (s) on the x axis and response units (RUs) on the y axis.

Figure S3

Cryo-EM analysis of CB6 Fab in complexed with prototype RBD-dimer and prototype-Beta chimeric RBD-dimer, respectively, related to Figure 1 (A–C) Cryo-EM analysis of CB6 Fab in complexed with prototype RBD-dimer. (D–F) Cryo-EM analysis of CB6 Fab in complexed with prototype-Beta chimeric RBD-dimer. (A and D) Flow chart of cryo-EM data processing for RBD-dimers bound to CB6. (B and E) Euler angle distribution of the final reconstruction. (C and F) The FSC curve for the reconstruction, the FSC 0.5 cutoff value is indicated by red dashed line.

Figure S4

Cryo-EM analysis of CB6 Fab in complexed with Delta-Omicron chimeric RBD-dimer and representative cryo-EM 2D class average images of RBD-dimers bound to CB6 Fabs, related to Figure 1 (A) Flow chart of cryo-EM data processing for Delta-Omicron chimeric RBD-dimer bound to CB6. (B) Euler angle distribution of the final reconstruction. (C) The FSC curve for the reconstruction, the FSC 0.5 cutoff value is indicated by red dashed line. (D) Representative cryo-EM 2D class average images of prototype RBD-dimer bound to two CB6 Fabs. (E) Representative cryo-EM 2D class average images of prototype-Beta chimeric RBD-dimer bound to one CB6 Fab. (F) Representative cryo-EM 2D class average images of Delta-Omicron chimeric RBD-dimer bound to one CB6 Fab.

Figure 2

Immunogenicity and protection efficacy of homotypic and prototype-Beta chimeric RBD-dimers Two batches of 8- to 10-week-old female BALB/c mice (n = 8 each batch) were immunized with two doses of 0.5-μg prototype, Beta or prototype-Beta chimeric RBD-dimer using AddaVax as adjuvant, 21 days apart. PBS plus adjuvant was given as the sham control. The first batch of BALB/c mice (n = 8) receiving two doses of vaccine or sham were challenged with Beta variant or prototype SARS-CoV-2 via the i.n. route at 46 and 61 days, respectively, post the second vaccination. (A) Sera collected at 14 days post the second immunization were tested for neutralization of a panel of pseudotyped viruses displaying prototype, Alpha, Beta, Gamma, Delta, Delta plus, Kappa, Lambda, and Omicron spikes. The values are the GMT ± 95% confidence interval (CI). The horizontal dashed line indicates the lower limit of detection (LLOD). (B) Radar plot demonstrating the neutralization profile of sera elicited by prototype, Beta or prototype-Beta chimeric vaccine against eight SARS-CoV-2 pseudotyped viruses. (C–H) Random selection of four mice in each group were challenged with 5 × 10⁵ TCID₅₀ of prototype SARS-CoV-2 (GISAID: EPI_ISL_514256-7) (C–E) and the other four were challenged with 1 × 10⁶ TCID₅₀ of Beta variant (GDPCC-nCoV84 strain) (F–H). Mice challenged with prototype SARS-CoV-2 had received Ad5-hACE2 intranasally 5 days before. (C) Pulmonary viral gRNA levels were detected by qRT-PCR. (D) Pulmonary viral sgRNA levels were detected by qRT-PCR. (E) Plots show correlations and corresponding two-sided p values between pVNT₅₀ of prototype SARS-CoV-2 and viral gRNA. (F) Pulmonary viral gRNA levels were detected by qRT-PCR. (G) Pulmonary viral sgRNA levels were detected by qRT-PCR. (H) Plots show correlations and corresponding two-sided p values between pVNT₅₀ of Beta variant and viral gRNA. For (C, D, F, and G), shown are the box and whiskers plots of 25th to 75th percentile with median as center and whiskers of minimum to maximum percentile. p values were analyzed with two-tailed unpaired t test (ns, p > 0.05; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001). For (E and H), red and gray lines indicate linear regression line and 95% CI, respectively. r and p values represent Spearman’s correlation coefficients and corresponding two-sided p values, respectively. Symbols represent individual mouse and may overlap for equal values. Horizontal dashed lines indicated the LLOD.

Figure S5

Evaluation of the immunogenicity and protection efficacy of prototype-Beta chimeric RBD-dimer vaccine in animals, related to Figures 2, 3, and 4 (A) Antisera from mice vaccinated with prototype, Beta or prototype-Beta chimeric RBD-dimer vaccines were tested neutralization of a panel of pseudotyped viruses displaying prototype, Alpha, Beta, Gamma, Delta, Delta plus, Kappa, Lambda and Omicron spike, respectively. (B) Rhesus monkeys (n = 12) were immunized with clinical-grade prototype-Beta chimeric vaccine (25 μg antigen + aluminum hydroxide adjuvant) or placebo (aluminum hydroxide adjuvant). Three doses were administrated on days 0, 21, and 42. These monkeys were bled for humoral immunogenicity evaluation before immunization and 14, 35, and 52 days after receiving the first dose. Blood was collected for cellular immune responses on day 48. At the days 56, 57, and 58, four prototype-Beta chimeric RBD-dimer-vaccinated macaques and four sham-vaccinated macaques were challenged with total 1 × 10⁶ TCID₅₀ prototype SARS-CoV-2, Beta variant and Delta variant, respectively. Nasal swabs, throat swabs and anal swabs were collected before challenging and at the days 1, 3, 5, 7 post infection for SARS-CoV-2 titration. Lungs were collected for virus titration and pathological examination on 7 DPI. (C) Antisera from vaccinated macaques were tested neutralization of a panel of pseudotyped viruses displaying prototype, Alpha, Beta, Delta and Omicron spike, respectively. (D) The macaques were challenged with prototype virus, Beta variant and Delta variant, respectively, and weight and temperature were monitored. The values are means ± SEM.

Figure S6

Histological pathology of lung sections of mice, related to Figures 2 and 5 (A) Shown are the typical lung sections from mice challenged with prototype SARS-CoV-2 or Beta variant by H&E staining. Scale bars, 100 μm. (B) Shown are the typical lung sections from mice challenged with SARS-CoV-2 Delta or Omicron variant by H&E staining. Scale bars, 100 μm.

Figure 3

Immunogenicity of prototype-Beta chimeric vaccine in rhesus macaques Twenty-four rhesus macaques (see Table S1) were immunized with 3 doses of prototype-Beta chimeric RBD-dimer vaccine or sham (n = 12), 21 days apart. Serum samples were collected according to the study schedule shown in Figure S5B. (A) Endpoint titer of antigen-binding IgG. (B) 50% neutralization titer of pseudotyped virus (prototype, Alpha, Beta, Delta, and Omicron) in serum. (C) 50% neutralization titer of authentic SARS-CoV-2 (prototype, Alpha, Beta, and Delta). (D) 50% neutralization titer of authentic SARS-CoV-2 (prototype, Beta and Omicron). The values shown in (A)–(D) are the GMT ± 95% confidence interval (CI). (E) Summed IFN-γ, IL-2 and IL-4 ELISPOT responses in PBMCs collected at day 6 after the third dose toward peptides spanning SARS-CoV-2 RBD. The values are means ± SEM. p values were analyzed with two-tailed Mann-Whitney test. (^∗∗p < 0.01; ^∗∗∗p <0.001; ^∗∗∗∗p < 0.0001).

Figure 4

Protection of rhesus macaques by prototype-Beta vaccine immunization (A, D, and G) Box and whiskers plots of 25th–75th percentile with median as center and whiskers of minimum to maximum percentile prototype (A), Beta variant (D), and Delta variant (G) viral gRNA in lung tissues at 7 DPI. Each dot represents a lung lope. The 28 lopes from four animals in each group were analyzed together. p values were analyzed with two-tailed Mann-Whitney test (^∗∗p < 0.01; ^∗∗∗∗p < 0.0001). (B, E, and H) Pooled analyses of pathological scores for lung tissues of macaques challenged with prototype SARS-CoV-2 (B), Beta (E), and Delta (H). The values are means ± SEM. p values were analyzed with two-tailed Mann-Whitney test (^∗∗p < 0.01; ^∗∗∗∗p < 0.0001). (C, F, and I) Histological pathology analyses of lung sections of macaques challenged with prototype SARS-CoV-2 (C), Beta (F), and Delta (I). Black bar represents 100 μm. (J–L) Box and whiskers plots of 25th–75th percentile with median as center and whiskers of minimum to maximum percentile viral gRNA at 0, 1, 3, 5, and 7 DPI in nasal swabs (J), throat swabs (K), and anal swab (L). Each dot represents an animal sample. The horizontal dash lines represent limit of detection, with the upper and lower ones for highest and lowest limit of detection, respectively.

Figure 5

Immunogenicity and protection efficacy of Delta-Omicron chimeric RBD-dimer vaccine Groups of 6- to 8-week-old female BALB/c mice (n = 10) were immunized with two doses of 2-μg prototype or Delta-Omicron chimeric RBD-dimer using AddaVax as adjuvant, 21 days apart. PBS plus adjuvant was given as the sham control. Sera collected at 14 days post the second immunization for antibody titration. (A) Mice sera were tested for neutralization of a panel of pseudotyped viruses displaying prototype, Alpha, Beta, Delta, Omicron (BA.1), and Omicron (BA.2) spike. The values are the GMT ± 95% confidence interval (CI). The horizontal dashed line indicates the lower limit of detection (LLOD). (B) Radar plot demonstrating the neutralization profile of sera elicited by prototype vaccine or Delta-Omicron chimeric vaccine against five SARS-CoV-2 pseudotyped viruses. (C–J) Random selection of five mice in each group were challenged with 6 × 10⁵ TCID₅₀ of Delta SARS-CoV-2 variant (CCPM-B-V-049-2105-8) (C–F) and the other five were challenged with 6 × 10⁵ TCID₅₀ of Omicron (BA.1) variant (CCPM-B-V-049-2112-18) at 58 days post the second immunization (G–J). Mice challenged with Delta variant had received Ad5-hACE2 intranasally 5 days before. (C) Pulmonary Delta viral gRNA levels were detected by qRT-PCR. (D) Pulmonary Delta viral sgRNA levels were detected by qRT-PCR. (E) Plots show correlations and corresponding two-sided p values between pVNT₅₀ of Delta variant and Delta viral gRNA. (F) Analyses of pathological scores for lung tissues of mice challenged with Delta variant. (G) Pulmonary Omicron viral gRNA levels were detected by qRT-PCR. (H) Pulmonary Omicron viral sgRNA levels were detected by qRT-PCR. (I) Plots show correlations and corresponding two-sided p values between pVNT₅₀ of Omicron variant and Omicron viral gRNA. (J) Analyses of pathological scores for lung tissues of mice challenged with Omicron variant. For (C, D, G, and H), shown are the box and whiskers plots of 25th–75th percentile with median as center and whiskers of minimum to maximum percentile. p values were analyzed with two-tailed unpaired t test (ns, p > 0.05; ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗∗p < 0.0001). For (E and I), red and gray lines indicate linear regression line and 95% CI, respectively. r and p values represent Spearman’s correlation coefficients and corresponding two-sided p values, respectively. Symbols represent individual mouse and may overlap for equal values. Horizontal dashed lines indicated the LLOD. For (F and J), the values are means ± SEM. p values were analyzed with two-tailed Mann-Whitney test (ns, p > 0.05; ^∗∗p < 0.01; ^∗∗∗∗p < 0.0001).