Fig. 4

Dose–response and Time-course Study of mRNA Transfection. The mRNA vaccine in-vitro expression levels over different doses and time points were measured using IVE-LC/MS/MS. HEK293T cells were dosed with a quadrivalent mRNA vaccine. Panel A presents the raw PRM data for the IVVDYMVQK peptide in response to varying doses. Panels B and C show the antigen peptide signal overlays at 24 h (in red) and 48 h (in blue) relative to specific HEK peptide signals. This is plotted against the total mRNA (in pmol) dosed per well for two representative HA antigens—Wisconsin HA in Panel B and Darwin HA in Panel C. Panels D and E illustrate the absolute amounts of Wisconsin and Darwin HA antigens in cell pellets relative to the total mRNA (in pmol) dosed per well. Panels F (R² = 0.77) and G (R² = 0.936) display the correlation between total protein per sample, measured by UV280 absorbance, and the average HEK peptide peak area at 24 and 48 h, respectively.