Figure 3.

RBD mRNA + CpG-CART generates early high levels of RBD neutralizing antibodies. BALB/c mice (n = 5) were immunized as described in Figure 2A. (A) Sera from mice immunized with RBD mRNA-CART (black), RBD mRNA + CpG-CART (blue), and CpG CART (orange) were collected on D28 and D60 and tested in a commercially available RBD-ACE-2 inhibition assay. The same set of serum samples was tested in a pseudotyped virus neutralization assay. RBD-expressing pseudovirus particles containing a zsGreen and firefly luciferase vectors were coincubated with titrated concentrations of heat-inactivated mouse serum. (B) The pseudovirus particle–serum mix was then added to wells containing ACE-2-overexpressing 293F cells. Firefly luciferase expression was measured at 48 and 72 h after the start of the experiment. On D60, BAL was harvested from RBD mRNA-CART (black), RBD mRNA + CpG-CART (blue), and CpG-CART (orange) immunized mice. (C) RBD-specific total IgG was assayed by ELISA. (D) BAL containing immunoglobulins was tested for their ability to inhibit binding of RBD to hACE-2 using a commercial ACE-2 inhibition kit. (E) Lung single-cell suspensions from naive mice (gray, n = 3) or mice vaccinated on D0 and D21 IV with 3 μg of RBD-mRNA + 3 μg of CpG (blue, n = 3) were collected on D28 and incubated with media alone, RBD protein, or an irrelevant protein (CD81-His) [5 μg/mL] for 48 h and stained for T cell activation markers CD134, CD137, and intracellular TNFα on CD4⁺ T cells. Data are shown as mean ± SD. Data representative of 3 independent experiments (B–D) and 1 experiment (E). * = P < 0.05, ** = P < 0.01 *** = P < 0.001, **** = P < 0.0001 one-way ANOVA (Tukey’s multiple comparison test) (D) or two-way ANOVA (Tukey’s multiple comparison test) (E).