Fig 1.
Construction and characterization of multivalent RBD mRNA vaccines.
a Schematic of the mRNA components of the vaccines. mRNAs encoded the signal peptide of human IgE followed by one or two copies of the receptor-binding domains (RBDs) of SARS-CoV-2 (Wuhan-Hu-1) or SARS-CoV (CUHK-W1) strains. A GFP-expressing vaccine was constructed as a control. b Western blot analysis of mRNA-encoded RBD protein expression. Each mRNA was in vitro transcribed and transfected into HEK293T cells for 24 h. Brefeldin A (5.0 μg/mL) was added to the cells at 8 h post-transfection to block protein secretion before cell lysis. Blots were probed with a rabbit anti-Spike antibody that recognizes both SARS-CoV and SARS-CoV-2 RBDs. GAPDH was probed as a loading control. c Distribution of RBD mRNA-LNP particle diameters measured by dynamic light scattering. For b and c, data from one experiment representative of three independent experiments are shown.
Fig 2.
Antibody responses in SARS-CoV and SARS-CoV-2 RBD mRNA-LNP-vaccinated mice.
a Experimental protocol. Groups of BALB/c mice (n = 6) were primed and boosted 2 weeks apart by intramuscular injection of 10 μg of each mRNA-LNP vaccine. Mice were bled on day 28 and sera were prepared. b–f Serum titers of SARS-CoV-2 Spike protein-specific (b, d–f) and SARS-CoV Spike protein-specific (c) IgG (b, c), IgG2a (d), and IgG1 (e). The ratio of IgG2a to IgG1 was calculated from the data presented in d and e. g, h Serum 50% neutralizing antibody titers (NT50) against SARS-CoV-2 (g) and SARS-CoV (h) pseudovirus infection of Vero cells. NT50 represents the titer required for 50% inhibition of maximal infection. Dotted lines indicate the limit of detection. ANOVA with Tukey’s multiple comparison test was performed for data analysis.*p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.
Fig 3.
Neutralizing activity against SARS-CoV-2 variants by sera from mice immunized with a heterodimeric RBD mRNA-LNP vaccine.
a Mice were primed and boosted with the SARS-CoV/SARS-CoV-2 heterodimeric RBD mRNA-LNP as described in Fig 2A and bled 4 weeks after first immunization. Direct binding IgG titers were assessed by ELISA against Wuhan-Hu-1 and B.1.351 Spike proteins. b–i Neutralizing antibody titers of sera from mice immunized with the RBD heterodimer mRNA-LNP (b-e), SARS_2-RBD monomer mRNA-LNP (f, g), and RBD homodimer mRNA-LNP (h, i) against infection by SARS-CoV-2 Wuhan-Hu-1 compared with Beta variant B.1.351 (b, f), Wuhan-N501Y (c), Wuhan-E484K (d), and Delta variant B.1.617.2 (e, g, i) pseudoviruses, and (j) The differences of neutralizing antibody titers in Group 2, 4 and 5 against Wuhan-Hu-1, Beta variant and Delta variant infection. Dotted lines indicate the limit of detection. Mann-Whitney test, ns, not significant.