(A) Critical binding residues for bNAbs based on relative binding to alanine-scanning mutants spanning the full H77 E1E2 sequence. Binding residues are marked with green spheres superimposed on the H77 E2 core structure (31). For reference, contact residues for mAb AR3C, identified by Kong et al., are indicated with blue spheres. Additional mAbs are shown in Supplemental Figure 3. In the table, critical binding or contact residues shared by at least two mAbs are highlighted in red, and those shared by all 4 mAbs in purple. (B and C) Competition binding between mAbs. The 6 most broadly neutralizing mAbs from s117 (blue) and s110 (green) are shown, with additional mAbs shown in Supplemental Figure 4. Relative binding of 2 μg/ml of the biotinylated mAbs to strain 1a53 E1E2 in the presence or absence of blocking mAbs at a concentration of 20 μg/ml. Combinations resulting in relative binding <0.7 or <0.35 are marked in yellow or red, respectively. (B) Competition binding between newly identified mAbs and each other. Values represent the average of two independent experiments performed in duplicate. (C) Competition binding between newly identified mAbs and reference bNAbs. Values represent the average of replicates from one experiment, except for AR3C, which was tested in duplicate in two independent experiments. (D) Clustering of the 6 most broadly neutralizing mAbs (blue or green) with reference bNAbs (red) based upon neutralization profiling. For each mAb, neutralization of each of 19 HCV pseudoparticles was measured, generating a neutralization profile, and pairwise Spearman correlations were measured between these neutralization profiles. Circles at each intersection were scaled by the magnitude of the correlation between the indicated mAbs. Hierarchical clustering analysis using these pairwise correlations is depicted as a tree. Numbers at tree nodes are approximately unbiased (AU) test values (49), indicating strength of support for a particular cluster.