Human Fc-Conjugated Receptor Binding Domain-Based Recombinant Subunit Vaccines with Short Linker Induce Potent Neutralizing Antibodies against Multiple SARS-CoV-2 Variants

Abstract

The coronavirus disease-19 (COVID-19) pandemic has been ongoing since December 2019, with more than 6.3 million deaths reported globally as of August 2022. Despite the success of several SARS-CoV-2 vaccines, the rise in variants, some of which are resistant to the effects of vaccination, highlights the need for a so-called pan-coronavirus (universal) vaccine. Here, we performed an immunogenicity comparison of prototype vaccines containing spike protein receptor-binding domain (RBD) residues 319-541, or spike protein regions S1, S2 and S fused to a histidine-tagged or human IgG1 Fc (hFC) fragment with either a longer (six residues) or shorter (three residues) linker. While all recombinant protein vaccines developed were effective in eliciting humoral immunity, the RBD-hFc vaccine was able to generate a potent neutralizing antibody response as well as a cellular immune response. We then compared the effects of recombinant protein length and linker size on immunogenicity in vivo. We found that a longer recombinant RBD protein (residues 319-583; RBD-Plus-hFc) containing a small alanine linker (AAA) was able to trigger long-lasting, high-titer neutralizing antibodies in mice. Finally, we evaluated cross-neutralization of wild-type and mutant RBD-Plus-hFc vaccines against wild-type, Alpha, Beta, Delta and Omicron SARS-CoV-2 variants. Significantly, at the same antigen dose, wild-type RBD-Plus-hFc immune sera induced broadly neutralizing antibodies against wild-type, Alpha, Beta, Delta and Omicron variants. Taken together, our findings provide valuable information for the continued development of recombinant protein-based SARS-CoV-2 vaccines and a basic foundation for booster vaccinations to avoid reinfection with SARS-CoV-2 variants.

Keywords: RBD; SARS-CoV-2 variants; recombinant protein subunit vaccines; spike protein.

Figure 1

Schematic diagram of recombinant subunit protein vaccines and experimental design. Expression vectors containing either S-His, S1-His, S1-hFc, S2-His, plus an RBD-His or RBD-hFc fusion protein (A) was transfected into HEK293 cells (B). Recombinant fusion proteins were purified by column chromatography (C) and resolved and visualized by SDS-PAGE (D) (Lane M: protein marker; Lane E: elution target protein). (E) Schematic diagram of murine immunization with S-His, S1-His, S1-hFc, S2-His, RBD-His and RBD-hFc vaccines. Female Balb/c mice were immunized twice with S-His, S1-His, S1-hFc, S2-His, RBD-His and RBD-hFc fusion proteins (n = 5 per group).

Figure 2

Serum-specific and neutralization antibody titers induced by recombinant vaccines. (A) Serum levels of IgG titer at 30 and 40 days after immunization were determined by ELISA. (B) Serum levels of IgG subtype titer at 30 and 40 days after immunization were determined by ELISA. (C) Serum levels of IgM titer at 30 and 40 days after immunization were determined by ELISA. (D) Neutralizing activity against SARS-CoV-2; data are presented as mean ± SD of absorbance at 450 nm (n = 5).

Figure 3

Mice antisera inhibited RBD binding to ACE2 and splenocytes of RBD-hFc immunized mice significantly increased cytokines secretion. (A) Inhibitions of RBD-His binding to cell-expressed ACE2 by RBD-hFc and S1-hFc mice antisera were measured by flow cytometry. (B) Inhibitions of RBD-hFc binding to cell-expressed ACE2 by RBD-His, S1-His, S2-His and S-His mice antisera were measured by flow cytometry. (C) Number of IL-4 secreting cells in splenocytes stimulated by recombinant subunit protein vaccines on day 40. (D) Number of IFN-γ secreting cells splenocytes stimulated by recombinant subunit protein vaccines on day 40. Data are presented as mean ± SD; *** p < 0.001.

Figure 4

SARS-CoV-2 RBD-hFc vaccine induced a protective cell-mediated immune response in mice. The RBD-hFc fusion vaccine increases populations of memory CD4+ (A) and CD8+ (B) T cells. *** p < 0.001.

Figure 5

Evaluation of linker size on immunogenicity. (A) Expression vector sequence containing SARS-CoV-2 wild-type, Alpha and Beta RBD sequence fused to hFc with a larger or small linker. (B) IgG titers in mouse sera were determined by ELISA. (C) Serum levels of IgM titer on days 21 and 42 were determined by ELISA. (D) Neutralizing activity in response to the SARS-CoV-2, alpha or beta variants, challenge. (E) Flow cytometry shows inhibition of recombinant protein binding to ACE2.

Figure 6

Cytokine and neutralizing antibody levels in immunized mice. IL-4, IL-6, TNF-α and IFN-γ levels in W2 (A) and S2 (B) mice were detected by ELISA. (C) Neutralization activities of W2 and S2 mice against wild-type, Alpha, Beta, Delta and Omicron variants were assayed by live virus. Data are presented as mean ± SD; ***, p < 0.001. All experiments were repeated at least twice.