Binding of the mAbs to escape mutant HAs.

<p>(A and B) Structures of the mutation sites. Amino acid mutation sites are mapped onto the ribbon structure of the H5 HA monomer (A) and a surface representation of trimeric H5 HA (B). The globular region is colored dark gray. Sites of escape mutations against AY-2C2 and OM-b mAbs are colored red, and those against YH-1A1 mAb are colored blue. (C and D) Results of flow cytometry. A series of pIRES-hrGFP-1α vectors expressing the mutant HA were transfected into 293T cells. At 24 hpt, the cells were collected and incubated with the OM-b (C), AY-2C2 (C), or YH-1A1 (D) mAbs, and the binding activities were determined by flow cytometry. The MFI of each mAb was normalized against that of the C179 mAb to account for different expression levels. The relative MFI of each mAb to that of the wild-type HA is shown. P-values were calculated by Student’s t-test. No significant differences with the wild type is denoted by n.s. Standard deviation is represented by vertical bars. (E) Surface of the D43N mutant. Surface maps of wild-type VN1194 HA (upper panel) and D43N mutant HA (lower panel) are shown. The mutant formation was simulated by MOE based on the crystal structure of the VN1194 HA monomer (orange, negatively charged area; white, neutral area; blue, positively charged area). (F) Surface of the G46E mutant. Surface maps of wild-type VN1194 HA (upper panel) and G46E mutant HA (lower panel) are shown. (G46 and E46 are shown in red).