S Trimer Derived from SARS-CoV-2 B.1.351 and B.1.618 Induced Effective Immune Response against Multiple SARS-CoV-2 Variants

Abstract

The spread of SARS-CoV-2 and its variants leads to a heavy burden on healthcare and the global economy, highlighting the need for developing vaccines that induce broad immunity against coronavirus. Here, we explored the immunogenicity of monovalent or bivalent spike (S) trimer subunit vaccines derived from SARS-CoV-2 B.1.351 (S1-2P) or/and B.1. 618 (S2-2P) in Balb/c mice. Both S1-2P and S2-2P elicited anti-spike antibody responses, and alum adjuvant induced higher levels of antibodies than Addavax adjuvant. The dose responses of the vaccines on immunogenicity were evaluated in vivo. A low dose of 5 μg monovalent recombinant protein or 2.5 μg bivalent vaccine triggered high-titer antibodies that showed cross-activity to Beta, Delta, and Gamma RBD in mice. The third immunization dose could boost (1.1 to 40.6 times) high levels of cross-binding antibodies and elicit high titers of neutralizing antibodies (64 to 1024) prototype, Beta, Delta, and Omicron variants. Furthermore, the vaccines were able to provoke a Th1-biased cellular immune response. Significantly, at the same antigen dose, S1-2P immune sera induced stronger broadly neutralizing antibodies against prototype, Beta, Delta, and Omicron variants compared to that induced by S2-2P. At the same time, the low dose of bivalent vaccine containing S2-2P and S1-2P (2.5 μg for each antigen) significantly improved the cross-neutralizing antibody responses. In conclusion, our results showed that monovalent S1-2P subunit vaccine or bivalent vaccine (S1-2P and S2-2P) induced potent humoral and cellular responses against multiple SARS-CoV-2 variants and provided valuable information for the development of recombinant protein-based SARS-CoV-2 vaccines that protect against emerging SARS-CoV-2 variants.

Keywords: SARS-CoV-2; beta variant; immune response; spike; trimer.

Figure 1

Design, expression, and purification of S-trimer. (a) Schematic representations of expression plasmids of SARS-CoV-2 spike protein S1-2P derived from B.1.351 and S2-2P derived from B.1.618. (b) WB analysis of purified S protein, HRP-conjugated anti-6× His antibody used as detection antibody. (c) Reducing SDS-PAGE analysis with Coomassie Blue staining of purified S protein. (d) Native-PAGE analysis with Coomassie Blue staining of purified S protein.

Figure 2

Humoral responses of immunized mice at 10 μg dose. (a) Schematic of mouse vaccination. S1-2P-specific IgG titer (b), S2-2P-specific IgG titer (c), E484K RBD-specific antibody titer (d), and Beta RBD-specific antibody titer (e) of immunized BALB/c mice were determined by ELISA. The dots of different colors represented mouse serum of different immune groups. The data were represented as the reciprocal of the serum dilution that led to a half-maximal binding signal. Statistical significance was determined using one-way ANOVA or t-test and is indicated as * for p < 0.05.

Figure 3

Cross-binding antibodies in the serum of vaccinated mice at different immunization doses. (a) Schematic of mouse vaccination. S1-2P-specific IgG titer (b), S2-2P-specific IgG titer (c), S1-D614G -specific IgG titer (d), E484K RBD-specific antibody titer (e), N501Y RBD-specific antibody titer (f), Beta RBD-specific antibody titer (g), Delta RBD-specific antibody titer (h), and Gamma RBD-specific antibody titer (i) of immunized BALB/c mice were determined by ELISA. The dots of different colors represented mouse serum of different immune groups. The data were represented as the reciprocal of the serum dilution that led to a half-maximal binding signal. Statistical significance was determined using one-way ANOVA or t-test and is indicated as * for p < 0.05.

Figure 4

Elevated cross-binding antibody responses after the third immunization. (a) The increased fold of serum antibody titer after the third immunization compared with that after the second immunization. S1-2P-specific IgG titer (b), S2-2P-specific IgG titer (c), S1-D614G -specific IgG titer (d), Omicron S trimer-specific IgG titer (e), E484K RBD-specific antibody titer (f), N501Y RBD-specific antibody titer (g), Beta RBD-specific antibody titer (h), Delta RBD-specific antibody titer (i), and Gamma RBD-specific antibody titer (j) of immunized BALB/c mice were determined by ELISA. The dots of different colors represented mouse serum of different immune groups. The data were represented as the reciprocal of the serum dilution that led to a half-maximal binding signal. Statistical significance was determined using one-way ANOVA or t-test and is indicated as * for p < 0.05.

Figure 5

Analysis of neutralizing antibodies in the serum of vaccinated mice. Neutralization titers of vaccinated mouse sera against SARS-CoV-2 prototype (a), Beta variant (b), Delta variant (c), and Omicron variant (d). Live SARS-CoV-2 virus neutralization was accessed in sera of vaccinated mice collected two weeks after the third immunization. The dots of different colors represented mouse serum of different immune groups. The data were represented as the reciprocal of the serum dilution that protected Vero-E6 cells from CPE compared with no serum control. Statistically significant differences were compared using one-way ANOVA or t-test and are indicated as * for p < 0.05 and ** for p < 0.01.

Figure 6

Spike-specific cellular immune responses. IL-2 (a), IFN-γ (b), Il-4 (c), and IL-5 (d) T cell responses of splenocytes from each mouse were shown as mean ± SEM for each animal. The dots of different colors represented mouse serum of different immune groups. Statistically significant differences were compared using one-way ANOVA or t-test and are indicated as * for p < 0.05.