CD40.CoV2-vaccinated animals survive SARS-CoV-2 infection and show neutralizing and cross-reactive antibody responses. (a) Design of the CD40.CoV2 vaccination strategy before SARS-CoV-2 infection. (b) Relative weight and survival of mock-vaccinated (grey) and vaccinated (blue) hCD40/K18-hACE2 transgenic mice after SARS-CoV-2 inoculation. Both parameters were recorded from days 0 to 12 post infection (pi). The mean ± SD is presented. A Mann-Whitney U test was conducted to compare differences in weight between the two groups on day 12 (n = 9–12 animals per group) (**P < 0.01). Kaplan-Meier survival curves were generated (n = 6–9 animals per group) and the P value was calculated using the [log-rank (Mantel-Cox) test] (*P < 0.05). (c) Viral load (genome equivalent/µg RNA) and viral infectious particle units (PFU/mg of tissue) in the lungs of mock-vaccinated (grey) and vaccinated (blue) hCD40/K18-hACE2 transgenic mice (n = 3 animals per group) on day 5 pi with the median plotted as a line. (d) Levels of IgG antibodies (AU) binding to Wuhan and VOCs SARS-CoV-2 RBD proteins before vaccination (baseline, -2 days post-vaccination (dpv), n = 9–12 animals per group), after the completion of the vaccination schedule (28 dpv, n = 9–12 animals per group), and 40 dpv (i.e., day 12 pi time point, n = 3–5 animals per group). (e) Levels of IgG antibodies (AU) binding to SARS-CoV-2 (circles) and SARS-CoV-1 (triangles) S proteins in mock-vaccinated (grey) and vaccinated (blue) animals at -2, 28, and 40 dpv. Medians [Min-Max] are shown. The grey dashed lines represent prime and boost vaccines. The red dashed line represents SARS-CoV-2 inoculation. Neutralizing activity of (f) anti-RBD antibodies (units/mL) and (g) anti-S antibodies (units/mL) in mock-vaccinated (grey) and vaccinated (blue) animals post-vaccination (open circles) and post-infection (solid circles). Medians ± Interquartile ranges (IQRs) are shown. Thirty plasma samples from unvaccinated mice were used to determine the threshold for positivity, defined as the whole units/mL value immediately above the concentration of the highest sample for RBD (i.e., 8 units/mL) and Spike (i.e., 4 units/mL) proteins. These results were reproduced in a second independent experiment (Figure S3).