Fig 1.
Design and characterization of the recombinant RBD-sFc-HR/trimer protein.
(A) Schematic view of the RBD-sFc-HR/trimer protein and the control protein RBD-HR/trimer. The RBD-sFc-HR/trimer includes an RBD (319–643 aa) sequence derived from wild-type SARS-CoV-2, the soluble monomeric immunoglobulin G 1 (IgG1) fragment crystallizable (sFc) and two heptapeptide repeats (HR1, amino acids L916–P1162; HR2, amino acids D1163–L1203) of the SARS-CoV-2 S2 subunit. (B) HPLC analysis of the recombinant RBD-sFc-HR/trimer and RBD-HR/trimer. The insets show the SDS-PAGE analyses of the eluted protein samples. mAU, milli-absorbance units; M, marker. (C) Biolayer interferometry (BLI) binding assay between the purified receptor ACE2 and RBD-sFc-trimer (left) or RBD-HR/trimer (right). (D) Enzyme-linked immunosorbent assays (ELISAs) for binding assays between our purified proteins (RBD-HR/trimer, RBD-sFc-HR/trimer, RBD-Fc, MSL109 (control)) and RBD-specific antibodies (CB6, n3113v, bn03). The data represent the means ± SDs.
Fig 2.
Extended half-life of the recombinant RBD-sFc-HR/trimer protein.
(A) For the RBD-sFc-HR/trimer or RBD-HR/trimer proteins, 5 mg/kg was intramuscularly injected, and the time points at which the serum was collected were recorded. Created with BioRender.com. (B) Pharmacokinetics of RBD-sFc-HR/trimer (blue) and RBD-HR/trimer (orange) in mice (n = 5), as measured by ELISA. The data represent the means ± SDs. (C) Schematic view of the RBD-sFc-HR/trimer protein binding to FcRn by ELISA. (D) ELISA for binding assays between RBD-HR/trimer and RBD-sFc-HR/trimer and FcRn at pH 6.0 and pH 7.4. The data represent the means ± SDs. (E) Schematic view of the RBD-sFc-HR/trimer protein binding to FcRn by BLI. (F) BLI binding assay between purified receptor FcRn and RBD-sFc-trimer or RBD-HR/trimer at pH 6.0 and pH 7.4.
Fig 3.
The RBD-sFc-HR/trimer vaccine elicited a potent humoral immune response and broad neutralization activity against SARS-CoV-2 variants.
(A) Schematic representation of the mouse immunization protocol. Serum samples were obtained from the mice at 14 d after each immunization. Created with BioRender.com. (B) Serum was collected on d 14 after the prime vaccination. The levels of IgM+IgG+IgA against the RBD protein were detected via a series of serum dilutions. The data represent the means ± SDs. (C) An assay of the endpoint titers of specific anti-RBD IgGs induced by our vaccine was performed via ELISA (n = 8 mice per group). (D) Serum was analyzed for IgG antibodies against recombinant RBDs from multiple variants of SARS-CoV-2 (Alpha, Beta, Delta, Lambda, Gamma, and Omicron) via ELISA (n = 8 mice per group). The absorbance at 405 nm was measured via a microplate reader with the wavelength correction set to 630 nm. (E) Neutralization of SARS-CoV-2 pseudovirus (wild-type or Omicron) infection in Huh-7 cells by immune serum (D 56). The data are presented as the means ± SDs. P values were determined via one-way ANOVA. A P-value < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).
Fig 4.
The RBD-sFc-HR/trimer vaccine induced durable neutralizing antibody responses and promoted GC B-cell generation.
(A) Schematic representation of the mouse immunization protocol. Serum samples were obtained from the mice at 14 d after each immunization. Created with BioRender.com. (B) Levels of specific anti-RBD IgGs at the endpoint titer on d 120. (C) Levels of specific anti-RBD IgG antibodies at the endpoint titer on d 162. The absorbance at 405 nm was measured via a microplate reader with the wavelength correction set to 630 nm. (D) Temporal profile of specific anti-RBD IgG levels from d 120 to d 162. (E-F) Neutralization of SARS-CoV-2 pseudovirus (wild-type or Omicron) infection in Huh-7 cells by immune serum (d 162). n = 8 mice in each group in B-E. (G) The frequency of germinal center B cells (CD3-CD19+GL7+CD95+) in the spleen (n = 5). The data are presented as the means ± SDs. P values were determined via one-way ANOVA. A p value < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).
Fig 5.
The RBD-sFc-HR/trimer vaccine elicited a robust T-cell response in vivo.
(A) The number of RBD-specific IL-4-producing memory T cells in the spleen was analyzed by flow cytometry after stimulation with the SARS-CoV-2 spike RBD peptide pool for three d. Memory T cells were gated on CD3+CD4+CD44+ (left) or CD3+CD8+CD44+ (right) populations. (B) The percentage of central memory (CD44+CD62L+) CD4+ or CD8+ T cells in the spleen. (C) The percentage of effector memory (CD44+CD62L-) CD4+ or CD8+ T cells in the spleen. n = 5 mice in each group in A-C. Spleen samples were obtained from the mice at 14 d after the third immunization. The data are presented as the means ± SEMs. P values were determined via one-way ANOVA. A p value < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).