Conceptualization, A.B., A.H.B. and S.D.; Funding acquisition, S.D.; Investigation, A.B., E.R., D.B., T.H., W.H., M.G., R.S.S., W.-H.C., E.J.M., J.L.J., V.D., L.M.-R., S.M. and J.K.; Resources, N.L.M., J.R., S.K., M.R., G.R.M., M.G.J., G.D.G. and S.D.; Writing—original draft, A.B., E.R., D.B. and S.D.; Writing—review and editing, A.B., E.R., W.-H.C., E.J.M., V.D., L.M.-R., N.L.M., S.K., M.R., G.R.M., M.G.J., G.D.G. and S.D. All authors have read and agreed to the published version of the manuscript.

Figure 1. An overview of the domain organization of SARS-CoV-2′s RBD and its purification from the E. coli expression system. (A) Domain architecture of the spike (S) protein of SARS-CoV-2 (not to scale). The subdomain 1 (S1) contains the N-terminal domain (NTD, a.a. 14−305) and the receptor binding domain (RBD, a.a. 319−542). The receptor binding motif (a.a. 438−506) is contained within the RBD. Subdomain 2 (S2) contains the fusion peptide (FP) and two heptad repeats (HR1, a.a. 908 −985; HR2, a.a 1163−1214), which are separated by the connecting domain (CD). The S protein is anchored within the viral membrane by a transmembrane membrane helix (TM); the intracellular domain (IC) remains inside the virus. The S1 subunit is released post-cleavage during the fusion process. (B) Expression and purification of Wuhan-Hu1 variant, (C) Beta variant, and (D) Alpha variant RBDs from E. coli. Lanes 1 & 2, −/+ IPTG: whole-cell bacterial lysates; lane 3, s/n: supernatant resulting from microfluidization and centrifugation; lane 4, pellet: pellet resulting from cell lysis (inclusion body); lane 5, inclusion body wash; lane 6, pellet resulting from urea solubilisation of inclusion body; lane 7, urea solubilized inclusion body (input for Ni-NTA); lane 8, FT, flow-through from Ni-NTA; lane 9, wash Ni-NTA; lane 10, elution Ni-NTA; lane 11, final RBD product post-dialysis.

Figure 1. An overview of the domain organization of SARS-CoV-2′s RBD and its purification from the E. coli expression system. (A) Domain architecture of the spike (S) protein of SARS-CoV-2 (not to scale). The subdomain 1 (S1) contains the N-terminal domain (NTD, a.a. 14−305) and the receptor binding domain (RBD, a.a. 319−542). The receptor binding motif (a.a. 438−506) is contained within the RBD. Subdomain 2 (S2) contains the fusion peptide (FP) and two heptad repeats (HR1, a.a. 908 −985; HR2, a.a 1163−1214), which are separated by the connecting domain (CD). The S protein is anchored within the viral membrane by a transmembrane membrane helix (TM); the intracellular domain (IC) remains inside the virus. The S1 subunit is released post-cleavage during the fusion process. (B) Expression and purification of Wuhan-Hu1 variant, (C) Beta variant, and (D) Alpha variant RBDs from E. coli. Lanes 1 & 2, −/+ IPTG: whole-cell bacterial lysates; lane 3, s/n: supernatant resulting from microfluidization and centrifugation; lane 4, pellet: pellet resulting from cell lysis (inclusion body); lane 5, inclusion body wash; lane 6, pellet resulting from urea solubilisation of inclusion body; lane 7, urea solubilized inclusion body (input for Ni-NTA); lane 8, FT, flow-through from Ni-NTA; lane 9, wash Ni-NTA; lane 10, elution Ni-NTA; lane 11, final RBD product post-dialysis.

Figure 2. Biochemical characterization of recombinant RBD. (A) SDS-PAGE analysis of E. coli- and mammalian-derived recombinant RBDs (~26 kDa). Lane 1: Wuhan-Hu1 variant; lane 2: Alpha variant; lane 3: Beta variant; lane 4: mammalian-expressed Beta variant (~30 kDa). (B) RBD functionality determined by association to the human-ACE2 receptor using BLI analysis. (C) Dot blot of E. coli- and mammalian-produced RBDs using the SARS-CoV-1 anti-RBD antibody, CR3022. Full-length P. falciparum CSP (FL-CSP) was used as the negative control. (D) Analytical size exclusion chromatography indicates that all purified RBDs were homogenous.

Figure 2. Biochemical characterization of recombinant RBD. (A) SDS-PAGE analysis of E. coli- and mammalian-derived recombinant RBDs (~26 kDa). Lane 1: Wuhan-Hu1 variant; lane 2: Alpha variant; lane 3: Beta variant; lane 4: mammalian-expressed Beta variant (~30 kDa). (B) RBD functionality determined by association to the human-ACE2 receptor using BLI analysis. (C) Dot blot of E. coli- and mammalian-produced RBDs using the SARS-CoV-1 anti-RBD antibody, CR3022. Full-length P. falciparum CSP (FL-CSP) was used as the negative control. (D) Analytical size exclusion chromatography indicates that all purified RBDs were homogenous.

Figure 3. Antigenicity of recombinant RBDs. Novel human mAbs produced against Wuhan-Hu1 (mAbs WRAIR-2057, -2063, -2123, -2125, -2151, -2165 and -2173) were tested for reactivity to recombinant Wuhan-Hu1, Alpha, and Beta variant RBDs from E. coli and the Beta variant RBD from mammalian cells using (A) BLI and (B) ELISA. MAbs WRAIR-2057, -2063, -2125, and -2151 showed broad reactivity, whereas mAbs WRAIR-2123, -2165, and -2173 were variant-specific. (C) Epitopes for WRAIR-2151 (PDB: 7N4M), WRAIR-2123, WRAIR-2125 (PDB 7N4L), WRAIR-2165, WRAIR-2173 (PDB: 7N4J), WRAIR-2057 (PDB: 7N4I), and WRAIR-2063 (PDB: 8EOO) are colored red, yellow, blue, pink, orange, green, and purple, respectively.

Figure 3. Antigenicity of recombinant RBDs. Novel human mAbs produced against Wuhan-Hu1 (mAbs WRAIR-2057, -2063, -2123, -2125, -2151, -2165 and -2173) were tested for reactivity to recombinant Wuhan-Hu1, Alpha, and Beta variant RBDs from E. coli and the Beta variant RBD from mammalian cells using (A) BLI and (B) ELISA. MAbs WRAIR-2057, -2063, -2125, and -2151 showed broad reactivity, whereas mAbs WRAIR-2123, -2165, and -2173 were variant-specific. (C) Epitopes for WRAIR-2151 (PDB: 7N4M), WRAIR-2123, WRAIR-2125 (PDB 7N4L), WRAIR-2165, WRAIR-2173 (PDB: 7N4J), WRAIR-2057 (PDB: 7N4I), and WRAIR-2063 (PDB: 8EOO) are colored red, yellow, blue, pink, orange, green, and purple, respectively.

Figure 4. Immunogenicity of recombinant RBDs. (A) Schematic of the mouse study design. (B) ELISA (OD = 1 titer) against Wuhan-Hu1, Alpha, and Beta coat antigens at 2WP3 (week 6) and at (C) 6WP3 (week 10) are shown. Individual mouse titers are indicated for each vaccine group (E. coli Wuhan-Hu1, black inverted triangles; E. coli Alpha, red circles; E. coli Beta, blue squares; trivalent, pink diamonds; mammalian Beta, green triangles), while geometric mean titers are indicated by each bar. (D) Inhibition of homologous RBD binding to human ACE2 by serum pool collected at week 10 (6WP3). Inhibitions are relative to inhibition by normal mouse serum at a 1:50 dilution (adjusted to 0%; not plotted). Significant p-values between groups are shown with * (p < 0.05).

Figure 4. Immunogenicity of recombinant RBDs. (A) Schematic of the mouse study design. (B) ELISA (OD = 1 titer) against Wuhan-Hu1, Alpha, and Beta coat antigens at 2WP3 (week 6) and at (C) 6WP3 (week 10) are shown. Individual mouse titers are indicated for each vaccine group (E. coli Wuhan-Hu1, black inverted triangles; E. coli Alpha, red circles; E. coli Beta, blue squares; trivalent, pink diamonds; mammalian Beta, green triangles), while geometric mean titers are indicated by each bar. (D) Inhibition of homologous RBD binding to human ACE2 by serum pool collected at week 10 (6WP3). Inhibitions are relative to inhibition by normal mouse serum at a 1:50 dilution (adjusted to 0%; not plotted). Significant p-values between groups are shown with * (p < 0.05).

Figure 5. Pseudovirus neutralization titers induced by vaccination with recombinant RBDs. Individual mouse ID50 titers are indicated for each vaccine group (E. coli Wuhan-Hu1, black inverted triangles; E. coli Alpha, red circles; E. coli Beta, blue squares; trivalent, pink diamonds; mammalian Beta, green triangles), while geometric mean titers are indicated by each bar. Mouse sera at week 6 (2WP3, top panels) and week 10 (6WP3, bottom panels) were tested in pseudovirus neutralization assays against Wuhan-Hu1 (A,B), Alpha (C,D), Beta (E,F), Delta (G,H), and Omicron BA.1 (I,J) pseudoviruses. Pre-immune sera showed ID50 titers <40 across all pseudoviruses (not plotted). Significant p-values between groups are shown with * (p < 0.05).

Figure 5. Pseudovirus neutralization titers induced by vaccination with recombinant RBDs. Individual mouse ID50 titers are indicated for each vaccine group (E. coli Wuhan-Hu1, black inverted triangles; E. coli Alpha, red circles; E. coli Beta, blue squares; trivalent, pink diamonds; mammalian Beta, green triangles), while geometric mean titers are indicated by each bar. Mouse sera at week 6 (2WP3, top panels) and week 10 (6WP3, bottom panels) were tested in pseudovirus neutralization assays against Wuhan-Hu1 (A,B), Alpha (C,D), Beta (E,F), Delta (G,H), and Omicron BA.1 (I,J) pseudoviruses. Pre-immune sera showed ID50 titers <40 across all pseudoviruses (not plotted). Significant p-values between groups are shown with * (p < 0.05).