Fig. 1: Vaccine design based on the receptor binding domain and transmembrane (TM) sequences.

a Schematic representation of the saRNA vector used for expressing RBD antigens. The saRNA vector consists of the VEE virus nonstructural polyproteins nsP1, nsP2, nsP3 and nsP4, a signal sequence (ss) and RBD or RBD fused to TM. b The Western blot images show RBD proteins detected in the media supernatant (Sup) and 293 T cells (Cell). Each lysate faction was harvested at 48 h post transfection. The experiment was performed at least 3 times and the representative image is shown. c BALB/c female mice (n = 5 per group) were injected intramuscularly three times with 107 IU/dose of replicon particle expressing SARS-CoV-2 Spike RBD at weekly intervals. Antibody titers of sera from the immunized mice were evaluated by ELISA against SARS-CoV-2 RBD proteins 1 week after the 2nd immunization (Day 14) and 3 weeks after the 3rd (Day 35). Plots represent individual endpoint titers with medians (lines). Mann–Whitney test (two-tailed) was used to determine significance. Experiment was performed two times independently. d The ACE2 and RBD binding inhibition titers of sera from the immunized mice (n = 5 per group) were measured. Plots indicate the –Log10 ID50 values with medians. Mann–Whitney test (two-tailed) was used to determine significance. Experiment was performed two times independently. e Schematic diagram of the antigen is showing the RBD (330-521aa) sequence (red), peptide linkers (327-329 aa) (blue), and (522-531 aa) (cyan). Black lines show the four disulfide bonds (Top panel). Structural models for the stabilized RBD as depicted in ribbon diagrams (Bottom panel) showing the location and structural orientation of the four disulfide bonds. RBD was modeled based on Protein Data Bank (PDB) code 6XM4 and displayed using PyMOL (http://www.pymol.org). Amino acids shown as pink and blue spheres are the cysteine formed disulfide bonds. f BALB/c female mice (n = 5 per group) were immunized intramuscularly twice at a four-week interval with 2.2 × 106 IU/dose of replicon particle expressing SARS-CoV-2 Spike RBD (original or stabilized) with TM. Antibody titers of sera from the immunized mice were determined using ELISA against SARS-CoV-2 Spike RBD protein. Plot represents individual endpoint titers with medians. One-way ANOVA was used to determine significance. Experiment was performed two times independently. g The ACE2 and RBD binding inhibition titers of sera from the immunized mice (n = 5 per group) were measured at 3 weeks after 2nd immunization (Day 42). Plots represent the –Log10 ID50 values with medians. One-way ANOVA was used to determine significance. Experiment was performed two times independently.