A Beta Strain-Based Spike Glycoprotein Vaccine Candidate Induces Broad Neutralization and Protection against SARS-CoV-2 Variants of Concern

Abstract

The coronavirus disease 2019 (COVID-19) pandemic is still ongoing. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) are circulating worldwide, making it resistant to existing vaccines and antiviral drugs. Therefore, the evaluation of variant-based expanded spectrum vaccines to optimize the immune response and provide broad protectiveness is very important. In this study, we expressed spike trimer protein (S-TM) based on the Beta variant in a GMP-grade workshop using CHO cells. Mice were immunized twice with S-TM protein combined with aluminum hydroxide (Al) and CpG Oligonucleotides (CpG) adjuvant to evaluate its safety and efficacy. BALB/c immunized with S-TM + Al + CpG induced high neutralizing antibody titers against the Wuhan-Hu-1 strain (wild-type, WT), the Beta and Delta variants, and even the Omicron variant. In addition, compared with the S-TM + Al group, the S-TM + Al + CpG group effectively induced a stronger Th1-biased cell immune response in mice. Furthermore, after the second immunization, H11-K18 hACE2 mice were well protected from challenge with the SARS-CoV-2 Beta strain, with a 100% survival rate. The virus load and pathological lesions in the lungs were significantly reduced, and no virus was detected in mouse brain tissue. Our vaccine candidate is practical and effective for current SARS-CoV-2 VOCs, which will support its further clinical development for potential sequential immune and primary immunization. IMPORTANCE Continuous emergence of adaptive mutations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to challenge the use and development of existing vaccines and drugs. The value of variant-based vaccines that are capable of inducing a higher and broader protection immune response against SARS-CoV-2 variants is currently being evaluated. This article shows that a recombinant prefusion spike protein based on a Beta variant was highly immunogenic and could induced a stronger Th1-biased cell immune response in mice and was effectively protective against challenge with the SARS-CoV-2 Beta variant. Importantly, this Beta-based SARS-CoV-2 vaccine could also offer a robust humoral immune response with effectively broad neutralization ability against the wild type and different variants of concern (VOCs): the Beta, Delta, and Omicron BA.1 variants. To date, the vaccine described here has been produced in a pilot scale (200L), and the development, filling process, and toxicological safety evaluation have also been completed, which provides a timely response to the emerging SARS-CoV-2 variants and vaccine development.

Keywords: SARS-CoV-2; broad neutralization; spike glycoprotein; variant-based vaccine; variants of concern.

FIG 1

Spike trimer protein (S-TM) design, expression, and characterization. (A) Linear diagram of the SARS-CoV-2 spike (S) protein ectodomain based on the protein sequence of Beta variant. S1 subunit has the N-terminal domain (NTD, dark blue) and receptor-binding domain (RBD, blue); S2 subunit has the fusion peptide (FP, purple), internal fusion peptide (IFP, black), heptad repeat 1 and heptad repeat 2 (HR1 and HR2, green), a flexible protein linker (GS), and a foldon sequence (red). The protein sequence mutations based on the Beta strain different from the WT strain were generated as follows: L18F, D80A, D215G, R246I, K417N, E484K, N501Y, D614G, A701V, R682Q, R683Q, R685Q, K986P, and V987P, with deletion of 242 to 244 amino acids. (B) SDS-PAGE analysis shows purified uncleaved S-TM protein expressed by CHO-K1 cells, with a molecular weight of around 180 kDa. (C) Negative-stain electron microscopy (EM) images of ectodomain of S-TM. (D) Two-dimensional images (performed by cisTEM) of S-TM show clear triangular protein particles.

FIG 2

Antibody induced upon immunization with S-TM based on the Beta variant. (A) Schematic representation of the study schedule. BALB/c mice (n = 8) were immunized with 1, 2.5, 5, 10, and 25 μg S-TM that was nonadjuvanted or adjuvanted with 50 μg Al or 50 μg aluminum hydroxide (Al) + 10 μg CpG twice on days 0 and 21 with primary analysis for humoral- and cell-mediated immunogenicity conducted on day 35 for blood and splenocyte samples. (B) S-TM binding antibody titers with ELISA. (C to E) Neutralizing antibody titers against Beta (C), wild-type (WT) (D), and Delta (E) strains with live SARS-CoV-2 assay. The points represent individual mice. For binding antibody (Ab), the bars represent the geometric mean with 95% CI of the antibody titer (EC₅₀), and comparisons were made using one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test. For the neutralizing antibody, the limitation of detection (LD) was initial dilution fold (1:25), data lower than the LD were considered initial dilution and were used as fold limited detection for calculation and plotting. Bars represent the geometric mean with 95% CI of the antibody titer, and the comparisons were made using ordinary one-way ANOVA multiple-comparison test. P values < 0.05 were considered significant. S-TM, spike trimer protein; CpG, CpG Oligonucleotides; MN50, the neutralization titer for 50% neutralization of viral infection; IFN, interferon; IL, interleukin; ns, not significant.

FIG 3

Cross-neutralizing based on live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralization titer after two doses of immunization with S-TM based on the Beta variant. (A) Schematic representation of the study schedule. BALB/c mice (n = 6) were immunized with 2.5, 5, and 10 μg S-TM that was adjuvanted with 50 μg Al + 50 μg CpG or only adjuvant without antigen twice on days 0 and 21; blood was collected at 14 days after the second vaccination for determination of neutralizing antibody titers. (B) Neutralizing antibody titer against the Beta, WT, Delta, and Omicron strains induced by S-TM + Al + CpG. (C to E) Neutralizing antibody titer changes among four virus strains in 2.5 μg (C), 5 μg (D), and 10 μg (E) groups. Points represent individual mice. Bars represent the geometric mean with 95% CI of the antibody titer, and comparisons were made using two-way ANOVA with Tukey’s multiple-comparison test. P values < 0.05 were considered significant. S-TM, spike trimer protein; Al, aluminum hydroxide; CpG, CpG Oligonucleotides; ns, not significant. GMT, geometric mean titer.

FIG 4

Cell-mediated immunity in different adjuvant group upon immunization with S-TM based on the Beta variant. BALB/c mice (n = 8) were immunized with 1, 2.5, 5, 10, and 25 μg S-TM that was nonadjuvanted or adjuvanted with 50 μg Al or 50 μg Al + 10 μg CpG twice on days 0 and 21 (different dose group was shown in Fig. S3). (A) The amount of immune cells in spleen secreting IL-2, IFN-γ, and IL-4 cytokines was determined, and the ratios of IFN-γ/IL-4 and IL-2/IL-4 were calculated 14 days after the second vaccination. (B) Frequency of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells in different groups. Bars indicate means ± standard error of the mean (SEM). Comparisons were made using one-way ANOVA with Tukey’s multiple-comparison test. P values < 0.05 were considered significant. S-TM, spike trimer protein; Al, aluminum hydroxide; CpG, CpG Oligonucleotides; SFU, spot-forming units; IL, interleukin; IFN, interferon.

FIG 5

Protection against SARS-CoV-2 Beta strain infection in H11-K18-hACE-2 mice after immunization with S-TM based on Beta variant. (A) Schematic representation of the challenge schedule. H11-K18-hACE-2 mice (n = 9) were immunized with S-TM + Al + CpG (10 μg) with or without adjuvants (only PB buffer) twice on days 0 and 21, and serum samples were taken at day 34 for immunogenicity assays. Two weeks after the second immunization, the mice were challenged with the SARS-CoV-2 Beta strain. Body weight was tracked, and the animals were euthanized for tissue sampling at 4 to 11 days postinfection (dpi). (B) Survival rates and changes in body weight after virus challenge in H11-K18-hACE-2 mice. (C) RNA copies in the lung and brain were detected by reverse transcription (RT)-PCR. The detection threshold (0.5 copies/μL) is shown by the dotted line in each panel. RNA copies below the threshold were set to 1/5 threshold for calculation and plotting. (D) Neutralizing antibody titer against the Beta, WT, and Delta strains 13 days after the second immunization. (E) Lung pathology scoring in mice at 4, 5, 8m or 11 dpi. For RNA copies and lung pathology scores, the bars indicate mean ± SEM, and statistical significance was calculated with two-way ANOVA with Tukey’s multiple-comparison test. For statistical analysis of antibody titers, the results are presented as the geometric mean with 95% confidence interval and statistical significance calculated with one-way ANOVA with Tukey’s multiple-comparison test. P values < 0.05 were considered significant. S-TM, spike trimer protein; Al, aluminum hydroxide; CpG, CpG Oligonucleotides; PB buffer: 20 mM Phosphate Buffer, 150 mM NaCl, and 0.02% polysorbate 80 (PS80), pH 6.

FIG 6

Histopathological analysis of SARS-CoV-2 Beta strain infection with or without S-TM-immunized H11-K18-hACE2 mice. The mice (n = 9) were immunized with S-TM + Al + CpG (10 μg) with or without adjuvants (only PB buffer) with two doses spaced 14 days apart. Two weeks after the second immunization, the mice were challenged with the SARS-CoV-2 Beta strain. Lungs were collected 4, 5, 8, or 11 dpi. The representative images of histopathology in lungs from the vaccinated group and the nonvaccinated control group are displayed. S-TM, spike trimer protein; Al, aluminum hydroxide; CpG, CpG Oligonucleotides; dpi, days postinfection; PB buffer: 20 mM Phosphate Buffer, 150 mM NaCl, and 0.02% polysorbate 80 (PS80), pH 6.