BALB/c mice (N = 5–6) were immunized twice with 10 μg mRNA/LNP vaccine or DS-Cav1 protein formulated with Adju-Phos®. Spleens were harvested 4 weeks following the second immunization. Splenocytes were incubated with pooled RSV F peptides and anti-mouse CD28 and were then stained with antibodies to the cell surface markers CD3, CD4, and CD8, as well as with a panel of anticytokine antibodies. The fluorescently tagged cells were then analyzed by flow cytometry using the gating strategy summarized in Supplementary Fig. 3. The percent of CD4+ T-cells (a) and CD8+ T-cells (b) responding to RSV F peptides with production of IFN-γ (white bar), IL-2 (light gray bar), or TNF-α (dark gray bar) is shown. Measurements for each animal are indicated (•). The height of the bar in the graph indicates the geometric mean calculation for the group ± 95% CI. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0001 compared with the DS-Cav1 protein group by two-sided unpaired T-test with Welch’s correction. The dark gray dotted line indicates the limit of detection of the assay.