A broadly generalizable stabilization strategy for sarbecovirus fusion machinery vaccines

Figures

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  Figure 1.. Design of prefusion-stabilized SARS-CoV-2 fusion machinery (S2 subunit) vaccines. A, Ribbon diagram of prefusion SARS-CoV-2 S highlighting all positions that were mutated to attempt to stabilize the metastable fusion machinery (S2 subunit) in the prefusion conformation. Mutations are shown in blue (intra-protomer disulfide bond), purple (VFLIP inter-protomer disulfide bond32), red (ten mutations selected based on expression/fusion score of a deep-mutational scan33), and green (subset of proline mutations selected from HexaPro31). The S1 subunit is shown as a transparent surface and glycans are omitted for clarity. B, Ribbon diagram of the C-44 cryoEM structure previously determined14 (PDB 8DYA). C, Size-exclusion chromatograms of the designed S2 constructs. D, Purification yields of the designed S2 constructs after size-exclusion chromatography. E, Ribbon diagram of the E-31 cryoEM structure. The position of the T961F mutation is circled red in one protomer. F, Superimposition of the S2 subunits from the E-69 cryoEM structure and prefusion SARS-CoV-2 S34 (gray, PDB 6XR8, residues 704–1146). The box denotes a local structural deviation. Glycans are omitted for clarity. G, Ribbon…

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  Figure 2.. A stable prefusion-stabilized SARS-CoV-2 fusion machinery (S2 subunit) vaccine candidate. A, Ribbon diagram of the E-69 prefusion-stabilized SARS-CoV-2 fusion machinery antigen highlighting the regions containing the epitopes recognized by S2 subunit-targeting monoclonal antibodies using dashed lines. B, Amino acid sequences of the RAY53, S2P6 and 76E1 epitopes. The V987P and D994Q E-69 mutations are respectively shown in green and red in panels A-B as they are located within the RAY53 epitope that has been previously reported24. C, Evaluation of retention of antigenicity for the E-69 antigen in various storage conditions using binding of the S2P6, 76E1 and RAY53 monoclonal antibodies analyzed by ELISA. D-H, Evaluation of retention of the native prefusion conformation for the E-69 antigen in various storage conditions analyzed by EM of negatively stained samples. Insets: 2D class averages. The scale bars represent 50 nm (micrograph) and 200 Å (2D class averages).

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  Figure 3.. A broadly generalizable prefusion-stabilization strategy for sarbecovirus fusion machinery (S2 subunit) antigens. A, Ribbon diagrams of superimposed S2 subunits of the prefusion SARS-CoV-2 S (PDB 6VXX34), SARS-CoV-1 S (PDB 5X5837) and PRD-0038 S (PDB 8U2936) structures. Prefusion-stabilizing mutations are shown in blue (intra-protomer disulfide bond), purple (VFLIP inter-protomer disulfide bond32), red (mutations ported from E-69), and green (subset of proline mutations selected from HexaPro31). B-I, Zoomed-in views of superimposed S2 subunits of the prefusion SARS-CoV-2, SARS-CoV-1 and PRD-0038 S structures highlighting the local structural conservation of residues mutated in SARS-CoV-2 E-69/F-53. Mutated residues in our designed constructs are underlined. SARS-CoV-2, SARS-CoV-1, and PRD-0038 S are respectively shown in light gray, gold, and pink in panels (A-I). J, Size-exclusion chromatograms of the designed SARS-CoV-1 and PRD-0038 S2 constructs, as compared to SARS-CoV-2 F53. K, Purification yields of the designed SARS-CoV-1 and PRD-0038 S2 constructs. The yield for the best SARS-CoV-2 S2 construct (F53) is included for comparison. L,M, Evaluation of retention of antigenicity…

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  Figure 4.. A prefusion-stabilized SARS-CoV-2 fusion machinery (S2 subunit) vaccine elicits broadly reactive antibody responses. A, Vaccination schedule and study design. B-D, Analysis of antibody binding titers against SARS-CoV-2 Hexapro S (B), XBB.1.5 Hexapro S (C), and SARS-CoV-1 Hexapro S (D) analyzed by ELISAs using sera obtained two weeks post dose 4. Geometric means and geometric SD are shown as bars. Representative graphs from two biological replicates are shown. E-G, Analysis of neutralizing antibody titers against SARS-CoV-2 Wu/G614 (E), XBB.1.5 (F), and SARS-CoV-1 (G) S VSV pseudoviruses using sera obtained two weeks post dose 4 expressed as area under the curve (AUC). Mean values and SD shown as bars. Baselines at 0 AUC represent no neutralization. Representative graphs from three biological replicates are shown.

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  Figure 5.. A prefusion-stabilized SARS-CoV-2 fusion machinery (S2 subunit) vaccine protects mice against SARS-CoV-2 XBB.1.5-induced disease. A, Weight loss followed 4 days post viral challenge with SARS-CoV-2 XBB.1.5 MA10. Control group represents the mice group without vaccination. Comparison of percent weight loss with the control group and each of the vaccinated groups was done using two-way analysis of variance (ANOVA) after Dunnett’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. B-C, Quantification of replicating viral titers in the nasal turbinates (B) and lungs (C) of challenged animals at 2 and 4 days post infection, respectively. Mean values and SEM shown as bars. Statistical significance compared with the control group and each of the vaccinated groups was reported using one-way ANOVA after Tukey’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Limit of detection shown as dashed line. D, Lung discoloration score at 2 and 4 days post infection. Mean values and SEM shown as bars. Statistical significance compared with the control group and each of the vaccinated groups was reported using one-way ANOVA after Dunnett’s…