FIG. 4.

Synergistic and synchronized antiviral activities of Fab bF-3674 and C34 further define the Fab 3674 epitope. (A) Dose response curves for antiviral activity against HXB2 Env-pseudotyped virus of C34 (green), bF-3674 Fab (blue), and a typical fixed combination ratio (1:10) of C34/bF-3674 (red). The experimental data (closed circles) are the averages for 6 to 10 independent experiments, with error bars indicating standard deviations and solid lines representing best-fit curves using the following simple activity relationship: percent fusion = 100/(1 + [Fab]/IC₅₀). The concentrations for the combination curve refer to the molar concentration of the C34 peptide. Dose reduction indices and CIs for different ratio combinations are provided in Table 4. (B) Synchronized viral infection assays using fully inhibitory concentrations of C34 or bF-3674 at increasing time points. Assays were run at 30°C (triangles) and 37°C (circles); data were fit to a sigmoidal curve as described previously (34). (C) Comparison of epitopes for neutralizing anti-gp41 antibodies Fab 3674 and D5. Mutagenesis and Western blot analyses show that Fab 3674-N-HR binding is governed by residues located at positions b and f of the heptad repeat (indicated in blue), while crystallographic analysis (27) shows that D5 binding to the N-HR occurs in a hydrophobic pocket located between two N helices. W571, located at position f, is critical for recognition of both antibodies.