Binding characteristics of coexisting IgG and IgA bNAbs. (A) Heatmap showing the ELISA binding of selected HIV-1 bNAbs to recombinant mutant, kifunensine-treated (gp120^kif), V3 loop-deleted (gp120ΔV3) gp120 proteins. Color value is proportional to the reactivity level measured as percentage of binding compared to WT gp120 in at least two independent experiments. (B) Representative ELISA graphs (top left) comparing the binding of selected antibodies from each identified bNAb clonotypes to WT and mutant proteins carrying sensitive substitutions. Means ± SD of duplicate OD_405nm values from two independent experiments are shown. Ribbon diagram showing the crystal structure of glycosylated gp120 subunit (gray; glycans in orange; PDB accession no. 5T3Z), in which putative contacting glycans of prototypical bNAbs are colored in blue. (C) Representative graphs showing the neutralizing activity of 7-269 IgA, 7-155 IgG, and control IgG bNAbs against YU2 pseudoviruses produced in the presence of kifunensine (YU2^kif; dotted lines) or not (YU2; straight lines). Means ± SD of duplicate values are shown. The heatmap (right) compares the IC₅₀ values (µg/ml) of the selected bNAbs against YU2 and YU2^kif. FC, fold-change. Mean values from two independent experiments are shown. (D) Competition ELISA graphs (left) comparing the binding of selected biotinylated bNAbs (-bio) in the presence of potential bNAb competitors. Means ± SD of duplicate OD_405nm values from two independent experiments are shown. Heatmap (right) showing competition for BG505 SOSIP.664 binding of selected HIV-1 bNAbs. Lighter colors indicate stronger inhibition; dark blue indicates no competition. (E) Binding of selected HIV-1 bNAbs to HEp2-expressing self-antigens as assayed by indirect IFA. Ctr+, positive control; Ctr− and ED38 are negative and low positive control antibodies, respectively. The scale bars represent 40 µm. (F) Microarray plots showing the reactivity of selected HIV-1 bNAbs to human proteins. Each spot corresponds to the z-scores given on a single protein by the reference antibody (Ref: mGO53, y axis) and test antibody (x axis). Red dots indicate immunoreactive proteins (z > 5) presented in Table S2. Frequency histograms in the upper left corner show the log₁₀ protein displacement (σ) of the MFI signals for HIV-1 bNAbs compared to nonreactive antibody mGO53. The PI corresponds to the Gaussian mean of all array protein displacements. (G) ELISA graphs comparing the binding to Env proteins of the selected bNAbs, GL, and mutated-GL hybrid counterparts. Means ± SD of triplicate OD_405nm values are shown (representative of three independent experiments). (H) In vitro neutralizing activity of mutated, GL, and hybrid versions of 7-269 bNAb. Dot plot (left) comparing the IC₅₀ values for the neutralization of clade B viruses (n = 5) as determined in the TZM-bl assay. 10-1074 and 7-269 are positive controls. Neutralization graph (right) shows the neutralizing activity of 7-269 and 7-269.IgL^GL against SC422661.8. Means ± SD of duplicate IC₅₀ values are shown (representative of three independent experiments).