Production of anti-gp140 HIV antibodies from single memory B cells.

A. Neutralization activity of purified IgGs from HIV-infected patients (pt9-11) sera measured by TZM-bl assay. The y-axis shows the IgG concentration required to achieve IC₅₀ for each viruses indicated on the x-axis: 1, MW965.26, 2, DJ263.8; 3, SF162.LS; 4, SS1196.1; 5, BaL26; 6, 6535,3; 7, RHPA4259.7; 8, SC422661.8; 9, TRO.11; 10, PVO.4 for pt9 and pt10, and CAAN5342.A2 for pt11; 11, YU2.DG. B. IgG antibody reactivity of pt9-11 patient sera against YU-2 trimeric gp140 and gp120 proteins determined by ELISA. The grey area indicates the reactivity range of the serum IgGs from previously studied clade B HIV-infected patients (pt1 to pt6) [25] used as comparison. C. Flow cytometry plots show the staining of patient PBMC with gp140 protein, anti-CD19 and anti-IgG antibodies. Gp140-rective IgG memory B cells were identified as gp140+IgG+CD19+ cells in the lymphocyte gate. The pie charts below the flow cytometry plots show for each patient the expansions of gp140-specific B-cell clones. The total number of memory B-cell antibodies is indicated in the center, each pie slice represents a clonal family and the area of the slice is proportional to the number of clonal relatives. Each clonal family is represented by the same color and unique antibodies that are not members of a clonal family are not colored. D. Gp140-antibody binding by ELISA of anti-gp140 monoclonal antibodies isolated from gp140+IgG+CD19+ cells in HIV-infected patients. The red and black dotted lines show positive (b12) [27] and negative (mGO53) [34] antibody controls. All the experiments were performed at least in duplicate. Representative data are shown. The pie chart indicates the numbers of gp140-reactive antibodies produced from each HIV+ donor with the total number in the center. E. Antibody binding to gp140 and gp120 measured by surface _lasmon resonance (SPR). The SPR sensorgrams for antibody binding to gp140 and gp120 overtime are shown for 10-939 and 10-923 as example. The antibodies were tested at concentrations ranging from 2.5 nM (black curve) to 40 nM (orange curve). RU, response units. F. Apparent affinities (K_A^app) to gp140 and gp120 ligands determined by SPR (as shown in E) for the anti-gp140 antibodies isolated from clade A HIV-infected African patients (filled circles) in comparison to the previously studied gp140-reactive antibodies produced from clade B HIV+ donors (open circles) [25]. Student t-test shows no statistical difference.