Unglycosylated Soluble SARS-CoV-2 Receptor Binding Domain (RBD) Produced in E. coli Combined with the Army Liposomal Formulation Containing QS21 (ALFQ) Elicits Neutralizing Antibodies against Mismatched Variants

Abstract

The emergence of novel potentially pandemic pathogens necessitates the rapid manufacture and deployment of effective, stable, and locally manufacturable vaccines on a global scale. In this study, the ability of the Escherichia coli expression system to produce the receptor binding domain (RBD) of the SARS-CoV-2 spike protein was evaluated. The RBD of the original Wuhan-Hu1 variant and of the Alpha and Beta variants of concern (VoC) were expressed in E. coli, and their biochemical and immunological profiles were compared to RBD produced in mammalian cells. The E. coli-produced RBD variants recapitulated the structural character of mammalian-expressed RBD and bound to human angiotensin converting enzyme (ACE2) receptor and a panel of neutralizing SARS-CoV-2 monoclonal antibodies. A pilot vaccination in mice with bacterial RBDs formulated with a novel liposomal adjuvant, Army Liposomal Formulation containing QS21 (ALFQ), induced polyclonal antibodies that inhibited RBD association to ACE2 in vitro and potently neutralized homologous and heterologous SARS-CoV-2 pseudoviruses. Although all vaccines induced neutralization of the non-vaccine Delta variant, only the Beta RBD vaccine produced in E. coli and mammalian cells effectively neutralized the Omicron BA.1 pseudovirus. These outcomes warrant further exploration of E. coli as an expression platform for non-glycosylated, soluble immunogens for future rapid response to emerging pandemic pathogens.

Keywords: E. coli; RBD; SARS-CoV-2; recombinant; vaccine.

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 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 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).