Fig 5.

Human anti-A56 MAbs have no effect on EV neutralization. (A to C) Vero E6 monolayers cell were infected with VACV-B5-GFP (green), and surface expression of A56 (red) was determined 12 h postinfection by surface staining with human anti-A56 MAb ES1 or WR2 and performing immunofluorescence (A) or flow cytometry (B and C). Surface expression of A56 was tested after infection with VACV-B5-GFP by surface staining infected cells with human anti-A56 MAb ES1 (red curve) or anti-DNP MAb (control; gray curve). (C) MFI of cell-based ELISA, quantitating surface-bound anti-A56 MAbs to VACV infected cells. Data are representative of three independent experiments. Irrelevant human MAb (anti-DNP, IgG1) was used as a negative control. (D) Sequence analysis of heavy-chain variable regions of human anti-A56 MAbs (WR2 and ES1). Framework (FR) and CDR regions are shown. (E) Human anti-A56 MAbs do not neutralize VACV EV. VACV EV neutralization activity of human anti-A56 MAbs (ES1 and WR2) with or without complement in the presence of anti-L1 Abs. Complement-fixing human anti-B5 MAb h101 (IgG1) was used as a positive control in each experiment. (F) Comet tail plaque inhibition. Shown are the absence of Ab (termed no treatment) or presence of anti-A56 MAbs (ES1 and WR2) or irrelevant MAb (control IgG1). Data are representative of two independent experiments. (G) Addition of complement-fixing anti-human IgG at 10 μg/ml (left) to human anti-A56 MAbs does not improve the neutralization of VACV EV. VACV EV neutralization activity of mouse anti-A33 MAb (NR565) in the absence or presence of complement-fixing anti-mouse IgG at 10 μg/ml with and without complement were control Abs (right). Irrelevant human MAb (anti-DNP, IgG1) was used as a negative control (right). The dashed line indicates 50% of the plaque numbers of VACV EV with or without complement. Error bars indicate SEM under each condition. All data in panels E and G are representative of three or more experiments.