Fig. 1
Development and characterization of an mRNA vaccine with broad-spectrum neutralizing protection against SARS-CoV-2 Omicron. a The mRNA construct of mRNA-D2-LNP expressing the SARS-CoV-2 RBD. b Delta RBD protein is expressed by mRNA in HEK293T cells. Cells were individually transfected with 4 mRNAs encoding Delta RBD (1 μg/mL) using Lipofectamine 2000 transfection reagent, and lysates were detected by Western blotting 16 hours after transfection. c Schematic diagram of mRNA-D2-LNP immunization, sample collection, and immunological assays. d, days; w, weeks; m, months. At days 10 and 24, Sera from immunized mice were assessed for Delta RBD-specific IgG antibodies (d) and pseudovirus-neutralizing antibodies (e) against Delta. After 5 months, Sera from immunized mice were assessed for Delta RBD-specific IgG antibodies (f) and pseudovirus-neutralizing antibodies (g) against Delta. ELISpot detection of IFN-γ (h) and IL-4 (i) release from SARS-CoV-2 RBD peptide stimulated splenocytes 56 days after mRNA-D2-LNP immunization. SFU = spot forming units. j Detection of SARS-CoV-2 RBD-specific CD4+ and CD8+ Tem cells (CD44+CD62L-) in splenocytes 5 months after mRNA-D2-LNP immunization by flow cytometry. k At day 24, Sera from immunized mice were assessed for pseudovirus-neutralizing antibodies against WT, Beta, Delta, BA.1, BA.2, and BA.4/5. l After 5 months, Sera from immunized mice were assessed for pseudovirus-neutralizing antibodies against WT, Beta, BA.1, BA.2, and BA.4/5. The mice were challenged intranasally with Omicron BA.1 (3.0 × 103 TCID50) at week 7 after immunization. Nasal turbinate (m) and lung (n) samples were collected 3 days after challenge to assess viral RNA load by RT-qPCR. o Stored at 4 °C mRNA-D2-LNP was measured particle size and PDI every ten days and particle size change were analyzed by GraphPad Prism 8.0. p The mRNA-D2-LNP was transfected in 293 T cells every 20 days (mRNA-D2 = 1 μg/ml) and the lysates were collected for Western blotting to detect the change in Delta RBD protein expression. The numbers are the mean of the corresponding groups. Data were analyzed by one-way ANOVA with multiple comparison tests. (no significant (ns) p > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001)
