Fig. 6: Effects of N1 NA tetramer stabilization on viral fitness and antigenicity.

a Growth curve of A/California 07/2009 H1N1 influenza reporter viruses with WT NA and NA with sNAP-155 mutations on MDCK-SIAT1-PB1 cells. Error bars denote the mean and error of the standard deviation (SD) of measurements from n = 64 individual wells at each time point. The experiment was repeated twice, and representative results are shown. b NS-EM of a recombinant soluble N1-CA09-sNAp-155 with the T466A mutation acquired during growth of the R3ΔPB1 A/California 07/2009 H1N1 sNAp-155 virus (scale bar, 10 nm). NS-EM preparation was performed once. c Models of Fabs of murine CD6 (purple) and human 1G01 (green) bound to CA09 NA. CD6 binds a quaternary epitope that spans two protomers, while 1G01 binds within and around the catalytic pocket. Models were generated by aligning crystal structures of CD6 (PDB ID 4QNP) and 1G01 (PDB ID 6Q23) Fabs to a single tetramer of CA09 NA (PDB ID 4B7Q). d Binding kinetics of anti-NA mAbs to N1-CA09-WT, N1-CA09-sNAp-130, N1-MI15-WT, and N1-MI15-sNAp-174. Biolayer interferometry sensorgrams of 1G01 IgG (left) and CD6 IgG (right) binding to N1-CA09-WT (red, top row), N1-CA09-sNAp-130 (blue, top row), N1-MI15-WT (red, bottom row), and N1-MI15-sNAp-174 (blue, bottom row). Upper and lower sensorgrams within each row were measured in buffers containing 1 mM CaCl₂ and 5 mM EDTA, respectively. Experimental data (colored traces) were fitted (black lines) with the binding equations describing a 2:1 interaction.