Fig. 1: Characterizing of engineered RBD constructs.
A Schematic of the 4RBD-Fc design. Four serially connected RBD (amino acid residues 319-591 of SARS-CoV-2 spike protein, green) were fused to a human IgG1 Fc domain at the C-terminal (yellow), with two 4RBD-Fc monomers forming a dimer. B The stability of RBD (319-591) and RBD (319-541) was compared by computing the backbone RMSD of the entire trajectories, carried out by VMD software. RMSD, root mean square deviation. C SDS-PAGE (left) of the eluted 4RBD-Fc samples, showing 4RBD-Fc assembly and purity. Size exclusion chromatography analysis of 4RBD-Fc (right). The ultraviolet absorption at 280 nm were shown. mAU, milli-absorbance units. M, marker; NR, non-reducing; R, reducing. D Representative raw images of 4RBD-Fc from negative-stain EM were shown. (Scale bar: 10 nm). Schematic of 4RBD-Fc dimer are shown to the right. E Competitive cell-surface binding of different RBD constructs. Relative binding (%) is calculated by measuring the reduction in mean fluorescence intensity (MFI) from RBD-FITC through flow cytometry. Kd values were calculated using the Cheng-Prusoff equation. Kd values were shown. F SARS-CoV-2 pseudovirus blocking assay. 293T-hACE2 cells were pre-incubated with serially diluted concentrations of RBD, 3RBD, or 4RBD-Fc and then infected with a luciferase-reporter SARS-CoV-2 pseudovirus. Blocking rate is calculated as the percent reduction in luminescence relative to control cells, which were not pre-incubated. G SARS-CoV-2 authentic virus blocking assay. VeroE6 cells were pre-incubated with 4RBD-Fc at indicated concentrations then infected with clinical isolated SARS-CoV-2 variants, the blocking rate is calculated as the percent reduction in viral load relative to control cells, measured by quantitative PCR with reverse transcription (qRT-PCR). Non-linear three-parameters inhibitor-response curve was used to determine the IC50 values. H BALB/c mice were i.m. immunized with RBD (n = 7), 3RBD (n = 7) or 4RBD-Fc (n = 12) as indicated and all adjuvanted with Alum and CpG, while mock group i.m. immunized with Alum and CpG only (n = 6), mice were sacrificed at 14 days-post immunization. I Serum was collected at day 14 for analysis, and anti-RBD IgG antibody titers were measured by ELISA. Endpoint titers were presented. J Neutralizing assay of day 14 serum in (H) use SARS-CoV-2 WT pseudovirus. IC50 was shown. Data are presented as mean ± s.d. The dashed line indicates the limit of detection (LOD). Undetectable values were set to LOD – 0.2 log units to distinguish them.
