Reactivity of AVFluIgG01 with different HA antigens.

<p><b>A</b>. Representative sequences of synthesized HA peptides (Viet04). <b>B</b>. Reactivity of AVFluIgG01 with the recombinant HA or HA1 proteins (Viet04) and overlapping peptides in ELISA. <b>C</b>. Reactivity of AVFluIgG01 with the native and reduced HA proteins (HK06) in ELISA. Synthesized peptides at 5 µg/ml or recombinant HA proteins at 1 µg/ml were used to coat 96-well microtiter plates, and AVFluIgG01 or a control mAb was tested at a final concentration of 10 µg/ml.</p

Inhibition of AVFluIgG01 on HA-mediated cell-cell fusion.

<p>A. AVFluIgG01 inhibits the low-pH induced cell-cell fusion in Vero cells that were transfected with the plasmid encoding Viet04 HA protein (pViet04-HA). The cells were stained with crystal violet. (a) Control cells (no HA transfection); (b) Positive control (without antibodies); (c) Treated with 50 µg/ml AVFluIgG01; (d) Treated with 50 µg/ml control mAb (AVFluIgG03). <b>B</b>. AVFluIgG01 inhibits cell fusion between 293T effector cells and MDCK target cells at a dose-dependent manner. 293T cells were co-transfected with pViet04-HA and pGAL4-VP16 and MDCK cells were transfected with pGal5-luc. After fusion induced by low-pH, the luciferase activity was measured in the cell lysates. The percent inhibition and IC₅₀ were calculated.</p

Inhibition of AVFluIgG01 on the pH-induced conformational changes of HA.

<p>The recombinant Viet04 HA proteins were incubated with or without AVFluIgG01 (<b>A</b>) or its Fab (<b>B</b>) at 37°C for 1 h. The mixture was either held at pH 7.5 (lanes 2, 4, 6 and 8) or changed to pH 5.0 (lanes 1, 3, 5 and 7) at 37°C for 1 h. After the pH was restored to 7.5, trypsin (1 µg/ml) was added to detect the acquisition of pH-induced protease susceptibility (lanes 5 to 8). The samples were then analyzed by western blotting. Molecular markers are given on the left.</p

Prediction of AVFluIgG01 epitope by minotopes.

<p><b>A</b>. Affinity-selected minotopes from a random peptide phage display library. The selection frequency of a specific minotope is shown in parentheses. <b>B</b>. Predicted amino acid residues by Mapitope. <b>C</b>. Predicted amino acid residues by Pep-3D-Search. The common residues that are predicted by both algorithms are underlined and shown in bold.</p

Mutagenesis analyses of the predicted epitope residues.

<p><b>A</b>. The reactivity of AVFluIgG01 with wild-type (WT) HA or HA containing site-directed mutations analyzed by flow cytometry. <b>B</b>. The reactivity of AVFluIgG01 with the WT (red curve) or HA carrying single mutations (green curve) analyzed by immuofluorescence assay (IFA). In both assays, the WT HA or mutants were transiently expressed on 293T cells by transfection and then immunostanined by AVFluIgG01 or a control mAb at a final concentration of 10 µg/ml.</p

Surface representation of the AVFluIgG01 epitope on the globular head of HA trimer model (PDB ID: 2IBX).

<p>Amino acid positions are designated in H5 numbering. <b>A</b>. Side view of the trimeric HA structure. The epitopic site I residues (I116, I117, P118) is colored in yellow, and site II (W122, S123) and site III (Y164, T167) residues are colored in blue and green, respectively. <b>B</b>. Top view of the globular head. The region encompassing the receptor binding site (RBS) is colored in red. <b>C</b>. Cartoon and sticks illustration of the three-dimensional structure of the AVFluIgG01 epitope. The diagram was generated with PyMOL.</p