Fig 1.
Schematic representation of pFUSE-hIgG1-Fc2 plasmid encoding RBD-Fc.
Fig 2.
Purification of RBD-Fc by an affinity and size exclusion chromatography.
(a) Elution profile of RBD-Fc using a mAbSelect column. (—) A280 was displayed as a solid line, while (—) conductivity was plotted as a dash line. (b) Size exclusion chromatogram (SEC) purification graph of RBD-Fc. A280 was plotted as a solid black line versus the retention volume.
Fig 3.
SDS-PAGE and Western blot analyses of purified RBD-Fc.
(a) Coomassie blue-stained SDS-PAGE analysis of non-reduced and reduced RBD-Fc. (b, c) Immunoblotting analysis of RBD-Fc using (b) anti-human Fc domain antibody and (c) anti-SARS-CoV-2 RBD antibody as detection antibodies in the presence and absence of DTT.
Fig 4.
Biochemical characterizations of recombinant RBD-Fc.
(a) Deglycosylation analysis of purified RBD-Fc using EndoH and PNGase F evaluated by SDS-PAGE followed by Coomassie-blue staining. (b) Dose-dependent binding of CHO-produced RBD-Fc (blue) and commercial RBD-Fc (red) to soluble human ACE2 as determined by ELISA. Data are presented as a mean of △OD450 ± S.D.
Fig 5.
Immunogenicity study and IgG subclasses profile of RBD-Fc formulated with different adjuvants in mice model.
(a) Schematic flow chart of the immunization experiment. Reciprocal anti-RBD (b) total IgG, (c) IgG1, and (d) IgG2a titers from mice sera collected in week 2, 5, and 8. Each dot represents a different subject. Limit of detection (titer less than 100) was presented in a dash line. The titers were presented as a geometric mean. (*, p<0.05; **, p<0.01; ***, p<0.001).
Fig 6.
Heat-map of NT50 titer of vaccinated mice sera against live SARS-CoV-2 virus.
Five BALB/C mice sera receiving different formulations of RBD-Fc vaccine were collected at two-week intervals after each immunization (week 2, 5, and 8) and tested by plaque reduction neutralization assay. Detection range is 100 to 6400.
Fig 7.
Calculated NT90 titer of mice sera against wild-type SARS-CoV-2.
Dilution of mice sera collected at week 5 and 8. Each dot represents an individual subject. The titers were displayed as a geometric mean. (*, p<0.05; **, p<0.01; ***, p<0.001).
Fig 8.
Inhibitory activity against interaction between SARS-CoV-2 RBD and host receptor, ACE2, elicited by immunized mice collected at week 8.
Mice sera were analyzed by competitive ACE2-coated ELISA against RBD conjugated with HRP. Titers were shown as a mice sera dilution that inhibited RBD-ACE2 interaction over 50% compared to negative control. Each dot represented individual subjects. The titers were plotted as a geometric mean. (*, p<0.05; **, p<0.01; ***, p<0.001).