Fig. 5: A mixture of four NA-F2A-HA mRNA-LNPs elicits a strong immune response to not only vaccine strain NA, HA, and HA stalk, but also diverse group 1 and 2 HAs, and limits disease following H1N1 viral challenge in ferrets.

a Schematic of vaccination and challenge experiment. Created in BioRender. (Heaton, N. (2024) BioRender.com/k70m759). b–e AUC analysis using cell-based ELISAs against 293Ts transfected with the NA or HA for the indicated virus strains. ELISAs are shown as net change in antibody reactivity between pre-vaccination sera (d-2) to post-vaccination (d28) sera. f–i PRNT₅₀ assays using the indicated viruses in MDCK cells. Any values under the LOD (ULD) (100, dotted line) are shown as 50 for inclusion in the plot. Any values over the LOD (OLD) (3200) are shown as 4000 for inclusion in the plot. j Change in bodyweight over 14 days post viral challenge. Dotted line = 0. k qRT-PCR on oral swab samples taken from ferrets at −3-, 2-, 4- or 7-days post infection. l Viral load determined from plaque assays using oral swabs taken from ferrets at −3, 2-, 4-, or 7-days post infection. Any values not detected (LOD = 10 PFU/mL, dotted line) are shown as 5 for inclusion in the plot. m, n Cell-based ELISAs (left) and corresponding AUC analysis (right) against 293Ts transfected with the indicated HA stalk expression plasmid. AUC analysis is shown as net change in antibody reactivity between pre- (dotted line) and post-vaccination (solid line) ferret sera. o Luminex binding assay of group 1 HAs (left), and group 2 HAs (right). Comparisons to the right of each panel are between the four mRNA-LNP- and Flulaval-vaccinated groups of animals. For all panels, n = 4 ferrets per experimental group and the experiment was performed once. mRNA-LNP vaccinated ferrets received 30 μg total of their respective vaccine (7.5 μg of each H1N1, H3N2, IBV-V and IBV-Y NA-F2A-HA LNPs for ‘four mRNA-LNP’ group), and Flulaval vaccinated ferrets received a full 60 μg of a Flulaval dose. Data are shown as means ± SEM. For all panels including statistics, significance was determined using Kruskal–Wallis test followed by pairwise Wilcoxon rank-sum tests with a Benjamini–Hochberg FDR correction. FDR-corrected p-values are reported above sample groups. Source data are provided as a Source Data file.