Capture of broadly HIV-1 neutralizing IgG and IgA memory B cell antibodies. (A) Radar plots comparing the in vitro neutralizing activity of IgG (red) and IgA (blue) antibodies purified from elite neutralizers’ sera against a five-virus panel. (B) Heatmaps showing the in vitro neutralizing activity (IC₅₀ in µg/ml) of purified serum IgG and IgA antibodies against two 12-virus reference panels as measured in the TZM-bl assay. (C) ELISA graphs (left) and flow cytometry plots (right) show the binding to BG505 SOSIP. 664 and YU2 foldon-type gp140 (gp140-F) trimers of purified serum IgG/IgA and IgG⁺/IgA⁺ memory B cells, respectively. OD_405nm, optical densities at 405 nm. Means ± SD of duplicate OD_405nm values are shown. (D) Pie charts (left) showing the distribution of unique vs. clonally expanded (Exp.) clones isolated by flow cytometric single B cell sorting using BG505 SOSIP.664 (top) and YU2 gp140-F (bottom) as baits. Colored slices indicate bNAb clonotypes for which dendrograms (right) show the relationship between clonally related IgH nucleotide sequences. Only antibodies highlighted in bold were expressed. (E) ELISA graphs comparing the reactivity of the selected HIV-1 bNAbs to recombinant Env proteins. Means ± SD of duplicate OD_405nm values are shown. (F) Heatmap showing the neutralization breadth and potencies of selected anti-gp160 IgG and IgA antibodies as measured in the TZM-bl assay. Means of duplicate values from two independent experiments are shown. Gray cells indicate nontested viruses with 7-319 only. (G) Heatmap comparing the antibody reactivity to Env proteins and neutralizing potencies of 7-107/7-269 clonal variants. (H) Neutralization coverage graph of 7-269 IgA bNAb against a panel of 56 viruses as measured in the TZM-bl assay. The y axis shows the cumulative frequency of IC₅₀ values up to the concentration shown on the x axis. The radar plot (upper left corner) shows the frequency distribution of neutralized viruses according to HIV-1 clades. (I) Coverage neutralization graph (left) comparing the breadth and potency of 7-269 IgA with 10-1074 and PGT121 IgG antibodies (historical data; Bouvin-Pley et al., 2014) in the TZM-bl assay against a clade B-virus panel (n = 38) covering three periods of the HIV-1 epidemic. Violin plots (middle) show neutralization IC₅₀ values for individual pseudotyped virus according to the periods of the epidemic. Pie charts (right) indicate the percentage of neutralized viruses by 7-269 per period. (J) Table presenting the immunoglobulin gene characteristics of the three bNAb clonotypes. n V_H Mut, number of somatic mutations in the V_H gene. (K) Bubble plot showing the percentage of clonal-related sequences (seq.) for the three pt7 bNAb lineages among all IgH sequences generated by Ig-HTS on DNA libraries from peripheral blood and bone marrow mononuclear cells, and filtered on bNAb-specific V-J rearrangements. (L) Representative graph showing the neutralizing (Neut.) activity of purified monomeric and dimeric 7-269 IgA antibodies (mIgA and dIgA, respectively) against YU2 pseudoviruses as measured in the in vitro TZM-bl assay. Means of duplicate values are shown. Heatmap (right) comparing the IC₅₀ values (nM) of 7-269 mIgA and dIgA antibodies against the selected HIV-1 strains. Means of duplicate values from two independent experiments are shown as in Fig. S2 C. FC, fold-change; norm, normalized values according to the number of antibody binding sites.