Fig. 1: Generation and characterization of H7-specific nanobodies.

a Schematic illustration of alpaca immunization process and subsequent nanobody selection. Alpacas were immunized with the H7N9 virus, and after four boosts, peripheral blood mononuclear cells (PBMCs) were collected to generate a phage display library. Created with BioRender.com. b Structural model of a nanobody-Fc created using ImmuneBuilder and AlphaFold 2. (Nanobody: orange. Fc tags: gray). c Immunofluorescence assay (IFA) showing the recognition of SZ19 H7N9 virus by different nanobodies. A549 cells were infected with SZ19 (MOI = 0.1) for 24 h and stained with primary nanobodies, followed by secondary goat anti-human IgG Fc Alexa Fluor™ 488 (A11: red, E10: orange, H10: yellow, F3: blue, H12: dark blue, C11: black.). Scale bar, 10 μm. d Western blot (WB) analysis of A549 cells infected with SZ19 H7N9 (MOI = 1) for 24 h. Cell lysate was probed with respective nanobodies and detected using goat anti-human IgG-Fc HRP. Data are representative of at least two independent experiments. e Graph showing 50% neutralization endpoint (NC₅₀) of different nanobodies against the SZ19 H7N9 virus. Virus was incubated with serially diluted nanobodies before cell infection. After 72 h, viral replication was measured by the ability of supernatant to hemagglutinate red blood cells (RBCs). Data are representative of at least two independent experiments. Shown are the mean values of three replicates. f Neutralization assay results for the nanobodies on SZ19 H7N9 virus using a cell-based ELISA. Half-maximal inhibitory concentrations (IC₅₀) are shown for each nanobody. Data represent the mean values ± SD from four technical replicates of three independent experiments. g Hemagglutination inhibition assay (HAI) with the nanobodies against SZ19 H7N9 virus (A11: red, E10: orange, H10: yellow, F3: blue, H12: dark blue, C11: black.). HI titer shown as µg/mL. Data are the average of three independent experiments.