Engineered SARS-CoV-2 receptor binding domain improves manufacturability in yeast and immunogenicity in mice

Abstract

Global containment of COVID-19 still requires accessible and affordable vaccines for low- and middle-income countries (LMICs). Recently approved vaccines provide needed interventions, albeit at prices that may limit their global access. Subunit vaccines based on recombinant proteins are suited for large-volume microbial manufacturing to yield billions of doses annually, minimizing their manufacturing cost. These types of vaccines are well-established, proven interventions with multiple safe and efficacious commercial examples. Many vaccine candidates of this type for SARS-CoV-2 rely on sequences containing the receptor-binding domain (RBD), which mediates viral entry to cells via ACE2. Here we report an engineered sequence variant of RBD that exhibits high-yield manufacturability, high-affinity binding to ACE2, and enhanced immunogenicity after a single dose in mice compared to the Wuhan-Hu-1 variant used in current vaccines. Antibodies raised against the engineered protein exhibited heterotypic binding to the RBD from two recently reported SARS-CoV-2 variants of concern (501Y.V1/V2). Presentation of the engineered RBD on a designed virus-like particle (VLP) also reduced weight loss in hamsters upon viral challenge.

Keywords: Pichia pastoris; SARS-CoV-2; manufacturability; protein vaccine.

Fig. 1.

Molecular engineering of the RBD for manufacturability. (A) Reduced SDS-PAGE of purified RBD. Sup, cultivation supernatant; Pur, purified protein. (B) Gene set enrichment analysis comparing strains expressing RBD and a rotavirus VP8 fragment (Left); schematic model based on pathways for degradation of the RBD in the proteasome and peroxisome, with higher flux of recombinant protein shown with larger arrows (Right). (C) Structural rendering of RBD (predicted hydrophobic patches are red). (D) Sequence logo of predicted ACE2 binding motif hydrophobic patch using the top 96 sequences homologous to SARS-CoV-2. Alignment of the ACE2 binding motif to other sarbecoviruses, including selected designs for testing. (E) Bar graph of relative specific productivity for engineered variants of the RBD. preOST1-proMF1 is an alternative signal peptide. Reported values are relative to expression of wild-type RBD. (F) Reduced SDS-PAGE of purified RBD-L452K-F490W. (G) Size exclusion chromatography of purified RBD variants. (H) Far-UV circular dichroism at 10 °C of purified RBD variants. (I) Differential scanning calorimetry of purified RBD variants. (J) Static light scattering vs. temperature of purified RBD variants.

Fig. 2.

Immunogenicity and antigenicity of wild-type and engineered RBD with single adjuvants. (A) Binding of purified RBD variants to human ACE2–IgG fusion protein and CR3022 neutralizing antibody by biolayer interferometry. (B) Titer of RBD-specific IgG in mouse sera by ELISA. Gray lines represent median values. (C) Titer of neutralizing antibody in mouse sera from SARS-CoV-2 pseudovirus neutralization assay. (D) Correlation of anti-RBD IgG ELISA and pseudovirus neutralization from mouse sera with SMNP adjuvant. (E) Titer of RBD-specific IgG in week 8 mouse sera from mice inoculated with RBD or RBD-L452K-F490W, evaluated for binding against RBD proteins with mutations from circulating strains of SARS-CoV-2. Data points represent individual animals. Gray lines represent median values. Significance was determined by t test, with Holm–Sidak correction. P values are indicated on plots. LOD, limit of detection.

Fig. 3.

Immunogenicity and antigenicity of engineered RBD nanoparticles in mice and hamsters. (A) Schematic of nanoparticle assembly using SpyTag and SpyCatcher. (B) Reduced SDS-PAGE of nanoparticle components. (C) Negative stain electron microscopy of SpyCatcher-12GS-I3-01 nanoparticles before (Left) and after (Right) conjugation to RBD-L452K-F490W-GGDGGDGGDGG-SpyTag. (D) Titer of spike protein–specific IgG in study 3 mouse sera by ELISA. Data points represent individual animals. (E) Spearman correlation of anti-S protein IgG ELISA and pseudovirus neutralization from week 5 mouse sera. (F) Titer of spike protein–specific IgG in hamster sera by ELISA. (G) Titer of neutralizing antibody in week 5 hamster sera by SARS-CoV-2 pseudovirus neutralization assay. (H) Mean percent body weight change of hamsters in each group after challenge with SARS-CoV-2. Error bars represent SEs. Gray bars represent median values. Significance was determined by t test. P values are indicated on plots.