Fig. 5: H7-HAK166T, S167L double mutant escapes E10 recognition but has lower viral fitness. | Nature Communications

Fig. 5: H7-HAK166T, S167L double mutant escapes E10 recognition but has lower viral fitness.

From: Influenza A Virus H7 nanobody recognizes a conserved immunodominant epitope on hemagglutinin head and confers heterosubtypic protection

Fig. 5

a IFA of A549 infected with wild-type (WT) or mutant (MUT) H7N9 virus (MOI = 0.1) at 24 h post-infection. Cell was stained with E10 as the primary antibody, followed by secondary goat anti-human IgG Fc Alexa Fluor™ 488. Nuclei were counterstained with DAPI (blue), and NP nanobody detection is shown in green. Data are representative of at least two independent experiments. b WB analysis of A549 cells infected with WT or MUT H7N9 viruses, with or without E10 pre-incubation, showing detection of NP and E10. Data are representative of at least two independent experiments. c Graph showing the 50% neutralization endpoint of E10 against WT and MUT viruses, measured by the ability of virus progeny to hemagglutinate red blood cells (RBCs). Data are representative of at least two independent experiments. Shown are the mean values of three technical replicates. d ELISA detection of WT-HA and MUT-HA hemagglutinin (HA) by E10 and F3 nanobodies. Bars represent mean ± SD. Data are representative of at least two independent experiments. Shown are the mean values of three technical replicates. e Experimental setup for the studies shown in (f, g). Created with BioRender.com. f Kaplan–Meier survival curve and body weight analysis of influenza-infected animals. Mice were treated intraperitoneally (i.p.) with either E10-Fc or PBS, as depicted in (e), and their weight was monitored for 14 days following infection with WT (106 EID50, lethal dose) or MUT virus (106 EID50). The statistical significance was calculated by log rank Mantel–Cox test for survival curve, p = 0.04. Weight change was monitored, and each graph is three experiments; n = 15; symbols represent means ± SEM. g Representative histopathological analysis of mouse lungs at day 3 post-infection with MUT virus, with or without E10 administration as outlined in (e). Scale bar, 500 μm in the left row, 200 μm in the middle row, 20 μm in the right row. h Growth kinetics of WT and MUT viruses in MDCK cells. Supernatants were collected at 6-, 12-, 24-, and 36-h post-infection (MOI = 0.001) and titrated by TCID50. Virus titers are presented as mean ± SD from three independent experiments. ***p = 0.001, ****p < 0.0001 (two-way ANOVA followed by Sidak test).

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