Fig 1.
Design and functional analysis of the glycosylated SARS-CoV-2 RBD.
(A) Design and construction of three mRNA expressing trimerization RBD with glycosylation. SP: signal peptide; T4f motif: T4 fibritin motif; (B) Circular dichroism of three purified RBD proteins; (C, D) Purified RBD proteins detected by SDS-PAGE and Western blot. (E) Western blot analysis of deglycosylated RBD proteins; (F) Purified RBD proteins detected by reducing and non-reducing page. The left line of each protein was subjected to 4–12% protein gel electrophoresis after boiling in 5×SDS-PAGE loading buffer, which disrupts the trimeric configuration, and the right line of each protein was subjected to 4–12% protein gel electrophoresis after mixed with 5×native sample loading buffer, preserving the protein’s oligomeric structure; (G) The affinity analysis of the monoclonal antibody B38 with different variants of RBD proteins and deglycosylated RBD. WT: wide-type, M1: Mutation 1, M2: Mutation 2.
Fig 2.
The encapsulation of mRNA vaccine and in vitro characterization.
(A) Schematic diagram of the LNP-mRNA vaccine; (B) Particle size analysis of LNP-mRNA vaccine before and after dialysis; (C) Particle size, PDI statistics, detection of encapsulation and loading rates of LNP-mRNA complexes; (D) TEM image of LNP-mRNA vaccine. Scale bar, 100 nm; (E) The pKa of LNP-RBDWT mRNA. (F) mRNA expression was detected after plasmid transcription in HEK-293T cells via qRT-PCR. (G) Proteins were expressed in HEK-293T cells followed determined by Western blot.
Fig 3.
Thermostability of LNP-mRNA formulations under different temperatures.
Fluc-encoding reporter LNP-mRNA was stored at 4°C, 25°C and 37°C for 1 d, 3 d, 7 d, and luciferase expression was detected by BLI in vivo and tissues in mice immunized i.m. for (A, B) 6 h (C, D) 24 h and (E, F) 48 h mice. SmG: submandibular gland.
Fig 4.
Humoral immune response in vaccinated mice.
(A) Schematic diagram of immunization, sample collection and antibody detection. Booster vaccination was administrated on days 14 (i), 21 (ii) and 28 (iii) after the primary immunization and serum was collected two weeks later. (B) Anti-SARS-CoV-2 wide-type RBD IgG titers. (C-H) Neutralization antibody of pseudovirus variants is shown with (C) Wuhan-Hu-1, (D) Beta, (E) Delta, (F) Omicron, BA.5 (G) SARS-CoV, (H) WIV1-CoV. (I-M) Neutralization antibodies against live SARS-CoV-2 Wuhan-Hu-1, Delta, Omicron BA.5, XBB and JN.1 strain. The black dashed line represents the limit of detection (LOD). Statistical significance was determined by one-way ANOVA and two-way ANOVA for comparisons of more than two groups with two or more items. (“*” represents p < 0.05, “**” represents p < 0.01, “***” represents p < 0.001, “****” represents p < 0.0001, “ns” represents not significant).
Fig 5.
Protection efficiency of the mRNA vaccines in a SARS-CoV-2 XBB challenge mouse model.
(A) Schematic of the experimental design, (B) Viral genome copies and SARS-CoV-2 titers in the lung at 3 d.p.i. were measured by qPCR. Statistical significance was determined by one-way ANOVA. (“*” represents p < 0.05, “**” represents p < 0.01, “ns” represents not significant).
Fig 6.
Analysis of RBD-mRNA immunized serum and splenocyte.
(A-C) ELISA analysis of RBDWT immunized serum on glycosylated and deglycosylated WT, M1, and M2 coated. (D-E) Antibody competition ELISA of RBDWT/M1/M2 immunized serum with WT and XBB as the coating antigen. (F) The representative IgG+ B cells from splenocyte after immunization are shown. Cells were gated for antigen-binding cells (IgG+ WT+ XBB+). The graph shows the percentage of double positive IgG cells for RBDWT and RBDXBB binding. Statistical significance was determined by one-way ANOVA or unpaired, two-sided Student’s t-test for two-group comparisons. (“*” represents p < 0.05, “**” represents p < 0.01, “ns” represents not significant).
Fig 7.
SARS-CoV-2-specific T cell immune response in mRNA vaccinated mice after stimulation of WT RBD-specific peptide pools.
(A-D) FCM analysis results show the percentages of CD4+ Tem cells secreting (A) TNF-α, (B) IL-2, (C) IFN-γ, and (D) IL-4. (E-H) FCM analysis results show the percentages of CD8+ Tem cells secreting (E) TNF-α, (F) IL-2, (G) IFN-γ, and (H) IL-4. Statistical significance was determined by one-way ANOVA. (“*” represents p < 0.05, “**” represents p < 0.01, “***” represents p < 0.001, “****” represents p < 0.0001).
Fig 8.
Humoral immune response in recombinant subunit protein-vaccinated mice.
(A) The immunization schedule for injection and sample collection. (B, C) Endpoint IgG titers against SARS-CoV-2 RBD were detected in serum collected at day 14 and 28 post-boost immunizations by ELISA. The adjuvant used in this study is the same volume Alum adjuvant, LOD = 100. (D-F) The 50% neutralization titer against live SARS-CoV-2 VOCs (Delta, Omicron BA.5, Omicron XBB strain). Serum was collected in 28 days post-booster immunizations. (G): Authentic neutralizing activity of monoclonal antibodies against SARS-CoV-2 and its variants. Statistical significance was determined by one-way ANOVA and two-way ANOVA for comparisons of more than two groups with two or more items. (“*” represents p < 0.05, “**” represents p < 0.01, “***” represents p < 0.001, “****” represents p < 0.0001).