Figure 1.

sa-mRNA bicistronic influenza A/H5N1 vaccine design and characterization in vitro

(A) Design schematic for subgenomic promoter (SGP) and internal ribosomal entry site (IRES) strategies. GOI, gene of interest; NSP, nonstructural protein; UTR, untranslated region. (B) Baby hamster kidney (BHK) cells were transfected with sa-mRNA bicistronic A/H5N1 vaccines and analyzed with flow cytometry for expression of hemagglutinin (HA) and neuraminidase (NA). Representative flow plots for cells expressing HA (X-axis) and NA (Y-axis) from different sa-mRNA bicistronic vaccines are labeled above the graph and the fraction of cells positive for both HA and NA are circled. Data were representative of at least three independent experiments. (C) Geometric mean fluorescence intensities (gMFI) for HA+ (left panels) and NA+ (right panels) cells for all tested sa-mRNA concentrations. Data were representative of at least three independent experiments. (D) BHK cells were transfected with sa-mRNA bicistronic A/H5N1 vaccines. Expression of NA and HA in cells was quantified by IDMS and results were normalized with GAPDH IDMS; the bar graph indicates the amount of relative antigen expression (fold difference) of the second GOI compared with the first GOI. Expression levels were averaged using four peptides for HA and three peptides for NA.