Extended Data Fig. 3: A significant conformational change to the HA stalk region induced by A388V mutation.

MDCK cells were infected with 1 multiplicity of infection (MOI) of wild-type (A388) or mutant (V388) H1N1pdm viruses generated by reverse genetics. 24 hours after infection, cells were harvested, and the expression of wild-type and mutant HA was measured by flow cytometry to evaluate the effect of the A388V mutation on the HA stalk epitopes. a, A representative example of the gating strategy. Dead cells and debris were excluded based on FSC/SSC cell dot plot. Anti-influenza nucleoprotein (NP) antibodies conjugated with allophycocyanin (APC) were used for gating. Only infected cells, expressing NP, were used for the analysis. Broadly neutralizing antibodies binding to the HA stalk, (b) CR6261, (c) CR9114, (d) FI6V3, (e) 70-1F02, (f) C179, and (g) CT149 were conjugated with fluorescein isothiocyanate (FITC). A monoclonal antibody that binds to the HA globular head, (h) EM-4C04, was conjugated with r-phycoerythrin (R-PE). Each stalk-binding antibody was mixed with the head-binding EM-4C04 antibody and NP antibody, and the antibody mixtures were used to stain cells expressing the wild-type or mutant HA. Histograms are colored differently to show different experimental groups: Blue - cells infected with wild-type (A388) virus; Pink - cells infected with mutant (V388) virus; Black - unstained cell control. Representative histograms from three independent experiments are shown. The summary table shows the average median fluorescence intensity (MFI) and standard error of mean (s.e.m.) of the three independent experiments.