Display of receptor-binding domain of SARS-CoV-2 Spike protein variants on the Saccharomyces cerevisiae cell surface

Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), represents a significant global human health threat. The most effective way to end the pandemic is through timely vaccination. In this study, the receptor-binding domains (RBDs) of Spike protein of the initial strain of SARS-CoV-2 and its variants, B.1.1.7 (Alpha), B.1.351 (Beta), and B.1.617.1 (Kappa), were successfully displayed on the surface of a Saccharomyces cerevisiae strain for development as a vaccine candidate. To rapidly express the recombinant protein and avoid the need for expensive galactose as an inducer, the gal80 gene of S. cerevisiae was knocked out, and the conventional 72-h culture period was thus successfully shortened to 24 h. Mice vaccinated against variant B.1.617.1 showed robust humoral and cellular immune responses. Moreover, the antiserum in the B.1.671.1 group had neutralizing activity against wild-type RBD and high binding titers against RBD mutants of variants B.1.351 and B.1.1.7. Double deglycosylation at N331Q and N343Q resulted in marked reduction of the affinity of RBD binding to angiotensin converting enzyme 2 (ACE2) and escaped antibody neutralization. This study demonstrates that yeast surface display technology can provide an alternative approach to rapid large-scale preparation of promising SARS-CoV-2 vaccine candidates at low cost.

Keywords: B.1.671.1; SARS-CoV-2; receptor-binding domain; vaccine; yeast surface display.

Figure 1

RBD displayed on the S. cerevisiae surface. (A) Schematic illustration of RBD displayed on the yeast surface. (B) Western blotting analysis of RBD and its mutant variants expressed on the surface of S. cerevisiae EBY200.

Figure 2

Immunofluorescence microscopy of recombinant yeast cells. (A) pYD1/EBY200. (B) pYD1-RBD (WT)/EBY200. (C) pYD1-mRBD (B.1.17)/EBY200. (D) pYD1-mRBD (B.1.617.1)/EBY200. (E) pYD1-mRBD (B.1.351)/EBY200 (left: light; right: matching fluorescence, Scale bar: 10 µm).

Figure 3

Determination of protein stability by immunofluorescence microscopy and ELISA. (A) pYD1-RBD/EBY200 before heat inactivation. (B) pYD1-RBD/EBY200 after heat inactivation. (C) Determination of protein stability of lyophilized yeast powder at 25°C for 1 month by ELISA. (D) Fresh lyophilized yeast powder at 4°C after 3 days. (E) Lyophilized yeast power at 4°C after 10 months. (F) Fresh lyophilized yeast powder with empty vector pYD1.

Figure 4

Humoral immune responses elicited by wild-type SARS-CoV-2 Spike RBD and its mutant variants. (A) Wild-type RBD-specific IgG serum antibody after 2nd (day 28), 3rd (day 44), and 4th (day 64) immunizations. (B) B.1.1.7, B.1.671.1, and B.1.1351 mRBD-specific IgG serum antibody after 4th (day 64) immunization. (C) The neutralization titer of B.1.617.1 mRBD antiserum against wild-type RBD was determined by competitive ELISA. Serum samples were collected 2 weeks after the fourth immunization (Day 64), and detected after mixing the samples from the same group in equal volume. Data points represent individual animals. For statistical analysis, t-test was performed to compare to PYD1/EBY200 (Empty vector) and PBS groups. Asterisks represent significance: *p < 0.05; **p < 0.01; n = 6. ns, no significant; ***: p< 0.001

Figure 5

Cellular immune responses in splenocytes from mice vaccinated with yeast surface-displayed B.1.617.1 mRBD. (A) The appearance of IFN-γ-expressing spots in an ELISpot assay plate. Each dot represents a single cell secreting IFN-γ. (B) Level of IFN-γ expression per 2 × 10⁵ splenocytes. (C) The total cytokine response on flow cytometry with intracellular cytokine staining 5 weeks after the fourth immunization or 120 days after priming. The levels of IFN-γ, IL-2, and TNF-α secretion by CD4⁺ and CD8⁺ T cells were quantified for each group and expressed as the frequency of cells expressing any one of the three cytokines. (D) Frequency of CD4⁺ T cells positive for each cytokine (IFN-γ, IL-2, and TNF-α) after stimulation with wild-type RBD antigen. (E) The proportions of CD4⁺ T cells secreting any combination of IFN-γ, IL-2, and TNF-α after stimulation with wild-type RBD antigen. A t-test was performed to compare the mRBD of the B.1.617.1 group and the PYD1/EBY200 (Empty vector) group. Asterisks represent significance: **p < 0.01; n = 6, ns, no significant; ***: p< 0.001.

Figure 6

Humoral and cellular immune responses of pYD1-RBD (wild-type)/EBY200 recombinant yeast cells inactivated with 25% ethanol at 40°C. Female BALB/c mice were subcutaneously immunized with recombinant yeast or PBS on days 1, 15, and 31. (A) Plasma samples collected on days 28 and 54 after initial immunization were used to determine the levels of RBD-specific IgG by ELISA. Data points represent individual animals. (B) Blood samples were collected on day 54 after initial immunization, and samples from the same group were pooled. Aliquots of 2 × 10⁵ PBMCs isolated from each pooled blood sample were stimulated with SARS-CoV-2 S-RBD antigen, concanavalin A (Con A), or medium only. IFN-γ-expressing cells were counted. Each dot represents a single cell that secretes IFN-γ. (C) Quantification of the number of IFN-γ-expressing cells per 2 × 10⁵ PBMCs. PBMC: peripheral blood mononuclear cell. For statistical analysis, t-test was performed to compare empty vector and PBS groups. Asterisks represent significance: **p < 0.01; n = 6, ns, no significant.

Figure 7

Glycosylation analysis of N331 and N343 of RBD. (A) Western blotting analysis of RBD protein and its mutant variants expressed on the surface of S. cerevisiae EBY200; lane 1: Mark, lane 2: PYD1-RBD/EBY200, lane 3: PYD1-RBD (N331Q)/EBY200, lane 4: PYD1-RBD (N343Q)/EBY200, lane 5: PYD1-RBD (N331Q&N343Q)/EBY200. (B) Binding of SARS-CoV-2 Spike protein RBD and its deglycosylation mutant variants to human ACE2. (C) Neutralizing activity of RBD and RBD (N331Q/N343Q) with SARS-CoV-2 IgG.