Design of the SARS-CoV-2 RBD vaccine antigen improves neutralizing antibody response

Abstract

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is the primary target of neutralizing antibodies and is a component of almost all current vaccines. Here, RBD immunogens were created with stabilizing amino acid changes that improve the neutralizing antibody response, as well as characteristics for production, storage, and distribution. A computational design and in vitro screening platform identified three improved immunogens, each with approximately nine amino acid changes relative to the native RBD sequence, and four key changes conserved between immunogens. The changes are adaptable to all vaccine platforms and compatible with mutations in emerging variants of concern. The immunogens elicit higher levels of neutralizing antibodies than native RBD, focus the immune response to structured neutralizing epitopes, and have increased production yields and thermostability. Incorporating these variant-independent amino acid changes in next-generation COVID vaccines may enhance the neutralizing antibody response and lead to longer duration and broader protection.

Fig. 1.. Overview of SPEEDesign pipeline used to create RBD immunogens.

(A) The SARS-CoV-2 spike trimer (green) binds human Ace2 (cyan) to mediate viral entry. This interaction is mediated by the up conformation of the RBD (gray), which can also exist in a down conformation (black). (B) The RBD design process retained the Ace2-interaction surface (cyan) and all known SARS neutralizing epitopes (blue). Residues exposed upon isolation of the RBD (green) were heavily designed, while all other residues (gray) were designed more conservatively. (C) Four computational strategies were used to create 40,000 decoys, each of which has an average of nine amino acid changes (red) from the native SARS-CoV-2 sequence. (D) Twenty-eight sequences sampling the top scoring decoys were screened in vitro, identifying five lead candidates (stars).

Fig. 2.. Stability and yield are higher for immunogens than WT RBD.

(A) All five immunogens expressed at higher levels than WT RBD and eluted as monomers by size exclusion chromatography (SEC). (B) SDS–polyacrylamide gel electrophoresis confirmed the high purity and yield of RBD immunogens. (C) Differential scanning fluorimetry indicated that four immunogens have higher thermostability than WT RBD. (D) T_m and purification yield averages and SDs from three separate purifications.

Fig. 3.. Structural basis for immunogen stabilization.

(A) Sequence alignment of amino acid changes in lead immunogens relative to the native RBD sequence. Four recurring changes are highlighted in pink. (B) Crystal structure of lead 1 (slate) in complex with scFv C144 (gray). (C) Crystal structure of lead 3 (green) in complex with Fab P2B-2F6 (gray). (D) The crystal structures of lead immunogens 1 (slate) and 3 (green) are globally similar to WT RBD (gray; PDB:7BWJ) despite amino acid changes (recurring, spheres; unique, sticks). Insets illustrate key recurring substitutions (pink).

Fig. 4.. Neutralizing antibody titers are higher in mice immunized with immunogens than WT RBD.

(A) Immunization and blood draw schedule for CD-1 mice. (B) Serum ELISA titers against trimeric FL-spike ectodomain. Dashed line indicates detection limit of assay, bars represent GMT, and error bars indicate geometric SD. (C) Titers of antibodies blocking Ace2/RBD interaction depicted as described in (B). (D) Pseudovirus neutralization titers depicted as described in (B). Statistical comparisons were made using a Kruskal-Wallis analysis of variance (ANOVA) followed by Dunn’s test corrected for multiple comparisons of immunogens and FL-spike with WT RBD. (E) Neutralization of WA-1 SARS-CoV-2 in a plaque assay. ID₅₀ values are indicated in parentheses. Serum from each animal in a group was pooled, except for the animal with the lowest pseudovirus neutralization titers in the FL-group, which had insufficient volume. (F) Antibody quality measured by ratio of pseudovirus neutralization titers (D) and FL-spike ELISA titers (B), depicted as described in (B). Statistical test is identical to (D).