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. 2023 Mar 5;26(4):106335. doi: 10.1016/j.isci.2023.106335

Figure 7.

Figure 7

Generation of EXG-5008, encoding RBD and nucleoproteins of SARS-CoV-2 and MERS

(A) A schematic diagram of EXG-5008, encoding a fusion protein of the signal peptide of CD5, the RBD and nucleoprotein of SARS-CoV-2, and the RBD and nucleoprotein of MERS-CoV.

(B and C) The frequency of IFN-γ spot-forming cells (B) and the frequency of IL-4 spot-forming cells (C) in 1 × 10ˆ6 splenocytes obtained from female C57BL/6 mice that were immunized by a single intradermal injection of 100 μL solution containing either placebo (PBO: buffer only), 25 μg of EXG-5006a, or 25 μg of EXG-5008. The splenocytes were restimulated by culturing in the presence or absence of pools of peptides: 15mer peptides for RBD of SARS-CoV-2 (original strain) (1); 15mer peptides for nucleoprotein of SARS-CoV-2 (original strain) (2); 15mer peptides for nucleoprotein of MERS-CoV (3); 15mer peptides for the spike of MERS-CoV (4). The cellular immunity was analyzed by ELISpot assays. The frequency obtained in the presence of peptides is plotted in the graph after subtracting the frequency obtained in the absence of peptides (background). The average and standard deviation (error bars) of five mice (n = 5) for PBO, four mice (n = 4) for EXG-5006, and five mice (n = 5) for EXG-5008, are shown for each group. Splenocytes were isolated 14 days after vaccination.

(D) A model showing how the c-srRNA booster vaccine works. Primary series of vaccines or prior infections expose naive B cells to viral surface proteins and turn them into memory B cells in a manner dependent on CD4+ helper T cells. Skin delivery of a c-srRNA booster vaccine encoding a fusion antigen of the viral surface proteins and internal proteins, whose sequences are more evolutionarily conserved than the surface proteins, is primarily incorporated into skin antigen-presenting cells such as Langerhans cells and dendritic cells. Within the antigen-presenting cells, c-srRNA replicates and produces the fusion antigen. The antigen-presenting cells digest the antigen into peptides and present these peptides to T cells. The peptides presented through this pathway stimulate MHC-I-restricted CD8+ cytotoxic T cells as well as MHC-II-restricted CD4+ helper T cells. CD8+ cytotoxic T cells eliminate infected cells (B) CD4+ helper T cells stimulate the memory B cells and enhance or restore the production of neutralizing antibody, which prevents infection.