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

Figure 3.

Figure 3

c-srRNA vaccines can prime the immune system for the subsequent induction of neutralizing antibodies against a variant antigen upon exposure to the variant antigen

(A) A schematic diagram of experimental procedures. EXG-5003 RNAs were prepared as naked RNAs, without LNP or other transfection reagents, in lactated Ringer’s solution.

(B–D) Groups of CD-1 outbred mice (N = 10) received one of three formulations by intradermal injection on Day 0 and Day 14 (black arrows): (B) a placebo (buffer only), (C) 5 μg of EXG-5003 RNA, or (D) 5 μg of EXG-5003 RNA in combination with an RNase inhibitor (3 units of RNasin Plus; Promega, Madison, WI). Subsequently, all mice received a recombinant RBD protein (Ala319-Phe541, with a C-terminal 6-His tag, NCBI accession number: YP_009724390.1, R&D Systems, Minneapolis, MN) by intradermal injection on Day 49 (open arrows). To assess humoral immunity, an enzyme-linked immunosorbent assay (ELISA), which quantitates the amount of immunoglobulin G (IgG) specific to a recombinant RBD protein (represented as the geometric mean of endpoint titer in triplicate), was performed on serum obtained from the mice on Day −3, Day 14, Day 28, Day 46, Day 56, and Day 63. Data are presented as mean (SD).

(E) A schematic diagram of experimental procedures. On day −40, blood was drawn from female BALB/c mice for a plaque reduction neutralization test (PRNT). On day −36, these mice received an intradermal injection of EXG-5003 RNA, which was prepared as a naked RNA, without an LNP or transfection reagent. On day −22 (14 days after EXG-5003 vaccination), half of the mice were sacrificed to obtain splenocytes for ELISpot assays. On day 0, the remaining mice were intradermally injected with the spike protein of the SARS-CoV-2 delta variant (B.1.617.2: R&D Systems) mixed with adjuvant—AddaVax (Invivogen). On day 7 (7 days after the spike protein injection), blood was drawn for the PRNT assay.

(F) The induction of cellular immunity against the RBD protein by a single intradermal administration of EXG-5003 RNA. The ELISpot assay shows the number of IFN-γ spot-forming cells (SFCs) in 1 × 10ˆ6 splenocytes from immunized mice restimulated by culturing in the presence or absence of a pool of 15mer peptides for RBD of SARS-CoV-2 (original strain). The number of SFC obtained in the presence of peptides was plotted on the graph after subtracting the number of SFC obtained in the absence of peptides (background). The average and standard deviation (error bars) of five mice (n = 5) are shown for each group.

(G) The titer of serum antibodies that can neutralize (50%) the SARS-CoV-2 virus (delta variant B.1.617.2), measured by the PRNT assay. Exposure to the spike protein of the SARS-CoV-2 virus (delta variant B.1.617.2) induced neutralization antibodies against the delta variant of the SARS-CoV-2 virus only in mice vaccinated with EXG-5003, which encodes the RBD of SARS-CoV-2 (original strain). Data are presented as mean (SD).