Fig. 1. SARS-CoV-2 RBD-Fc Vacc induced a protective immune response in nonhuman primates and mice.

a Two RBD domains were fused through the Fc fragment to form the Y-shaped structure via protein structure prediction server version 3.0 (left panel). The RBD-Fc protein expressed in CHO cells was identified by western blot analysis under reducing and nonreducing conditions using both antiserum from recovered COVID-19 patients and a commercial antibody (right panel). b N-glycosylation and O-glycosylation sites were identified by mass spectrometry. Docking between ACE-2 and RBD-Fc was predicted by the ZDOCK server. An overview of the glycosylation sites is shown based on the solved complex structure of SARS-CoV-2 RBD-Fc bound to ACE2. The identified sites, red for N-glycosylation and purple for O-glycosylation, are shown as spheres and labeled. The right panel (surface representation) was generated by rotating the structure in the left panel (cartoon representation) around a vertical axis by approximately 90° (lower panel). c Schematic diagram of immunization, sample collection and challenge schedule. d Macaca fascicularis macaques (n = 5) were immunized on days 0, 14, and 28 with 20 µg and 40 µg doses of RBD-Fc Vacc or with PBS, and serum was collected at the indicated times. The level of SARS-CoV-2 RBD-specific IgG was examined by ELISA. e Neutralizing antibodies were determined by a microneutralization assay using SARS-CoV-2 (NT50). f Viral load of nasal specimens collected from the inoculated immunized Macaca fascicularis (n = 4) on day 2, day 4 and day 6 post infection were monitored. g Viral loads in tissue from various lobes of the lung in inoculated Macaca fascicularis macaques (n = 4) on day 7 post infection were measured. h Histopathological analysis of lungs from inoculated Macaca fascicularis macaques (n = 4) on day 7 post injection. Lung tissues were collected and stained with hematoxylin and eosin. i hACE2-Tg mice were immunized three times as described in the materials and methods (n = 8). Postinfection viral loads in lung tissue from inoculated hACE2-Tg mice were measured. The dashed lines indicate the detection limit of the assay. j Histopathological analysis of lungs from all inoculated hACE2-Tg mice post injection. k Correlations of SARS-CoV-2 NAb titers (left panel), pseudovirus NAb titers (center panel), and RBD protein-specific IgG titers (right panel) in hACE2-Tg mice prior to challenge with an amount of virus equivalent to the log peak mRNA copies/g in lungs measured after challenge. The red lines indicate the best-fit relationship between these variables. The P and R values were calculated by two-sided Spearman rank correlation tests. In d)-k), all data are presented as the mean ± SEM of two independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001