Journal: Scientific Reports
Article Title: Influenza A virus derived NS1 enhances translation of HPLC purified mRNA and interferon adjuvanted mRNA vaccination
doi: 10.1038/s41598-026-35611-5
Figure Lengend Snippet: ( A ) C2C12 cells (n=5) were seeded in 24 well plates and co-transfected with Ψ modified mRNAs encoding luciferase (L) , GFP (G) and PR8S (P) at mass ratios L G P = 1 : (1−ε): ε (where ε is 0, 0.5 or 1) . 4 h after mRNA complexes were added, cells were treated with cytokines supplemented media at non-toxic concentrations IFN-β (50ng/mL), IL-1β (1ng/mL) or IL-6 (1ng/mL) for 18 h followed by luciferase assay. Cell viability of all groups were similar (Supplementary Figure S5). Results were normalized by luciferase expression of non-treated cells and reported as relative luciferase expression representing extent of translation inhibition by cytokines. Results were presented as the mean SEM. ( B ) C2C12 cells (n=5) were co-transfected with Ψ modified mRNAs encoding luciferase (L), IFN-α (I), GFP (G) and PR8S (P) at mass ratios L I G P = 2 : 1 : (1−ε): ε (where ε is 0, 0.5, 0.75 or 1). Control cells for each mass ratio were similarly transfected with G replacing I i.e. L G G P = 2 : 1: (1−ε) : ε. Luciferase expression was measured 18h later. Cell viability of all groups were similar (Supplementary Figure S5). Results were presented as the mean SEM. ( C ) C57Bl/6 Mice (n=8) were intramuscularly injected with 8 µg Luc mRNA, together with 8 µg GFP mRNA (Luc+GFP), 4 µg GFP mRNA + 4 µg IFN-α mRNA (Luc+GFP+IFN), 4 µg GFP mRNA + 4 µg PR8S mRNA (Luc+PR8S+GFP), or 4 µg PR8S mRNA + 4 µg IFN-α mRNA (Luc+PR8S+IFN). Non-treated (NT) group was injected with buffer without mRNA. Bioluminescence was assayed by IVIS 5 h after injection and once daily thereafter. Results were presented as the mean SEM. ( D ) C57Bl/6 Mice (n=4) were intramuscularly injected twice (7 days apart) with 8 µg OVA mRNA, together with either 8 µg GFP mRNA (OVA+GFP), 4 µg GFP mRNA + 4 µg IFN-α mRNA (OVA+GFP+IFN), 4 µg PR8S mRNA + 4 µg GFP mRNA (OVA+PR8S+GFP), 4 µg PR8S mRNA + 4 µg IFN-α mRNA (OVA+PR8S+IFN). Non-treated (NT) group was injected with buffer without mRNA. 7 days after the second injection, blood was collected by cardiac puncture and anti-OVA IgG titers were quantified by ELISA. Results were presented as the mean SEM with individual values shown.
Article Snippet: NIH-3T3 mouse fibroblasts, C2C12 mouse myoblast cell line and HepG2 liver cancer cells were purchased from the American Type Culture Collection (ATCC); NIH-3T3 cells were cultured in Dulbecco’s Modified Eagle Medium (Hyclone).
Techniques: Transfection, Modification, Luciferase, Expressing, Inhibition, Control, Injection, Enzyme-linked Immunosorbent Assay
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