Competitive binding assays by human serum samples. (A) Inhibition of MIP1β binding to CD4⁺ T cells in sera samples of a pool of 6 HIV-exposed seronegative (ESN), 207 unexposed seronegative (HC), 85 HIV⁺ seropositive LTNP (20 competing and 65 not competing), 135 chronically HIV-infected (HIV+HAART), and 70 progressor(P) patients. The values are expressed as the percentage of inhibition of MIP1β binding and are representative of 3 experiments performed. Error bars represent SD of 5 replicates per each data point. The cutoff value was set at 12% (3 SDs above the mean value of the HC serum samples, as previously described³⁶ ). • indicates mean value for each subject; ♦, mean value of each group. (B) Competitive binding assays comparing the 20 LTNP sera displaying competitive activity against MIP1β and CCR5. Abs were affinity purified on Sepharose column, then quantified by ELISA, and tested in MIP1β binding inhibition. Ig-enriched and Ig-depleted fractions from HCs were used as controls. The data are expressed as a percentage of MIP1β binding inhibition to CD4⁺ T cells and are representative of 3 experiments performed. Error bars represent SDs of 5 replicates per data point. (C) Dose-response curves of Ig-enriched fractions mediated binding inhibition of MIP1β binding to CD4⁺ T cells. Serum samples from 11 LTNP MIP1β competing and from a pool of 5 HCs were affinity purified by Sepharose column. Ig-enriched fractions were quantified by ELISA. The data are representative of 2 experiments performed. (D) MCP1 binding assay inhibition was carried out on CCR2-expressing transfected U87 cell line and CD4⁺ T lymphocytes from HCs. The data are representative of 3 experiments performed. Error bars represent SDs of 5 replicates per each data point. MCP1 cold and Igs from HCs were used as positive and negative controls, respectively. (E) Binding of pep 3 purified Igs to either CCR5- or CXCR4-expressing transfected U87 cell line. Peptide 3 corresponds to the second external domain of CCR5 as shown in Table 1. Total serum Abs were purified on Sepharose column and quantified by ELISA, and then Ig fractions were affinity purified on the relative CCR5 peptide as described in “Materials and methods.” A pool of serum Igs from the HCs was used as a negative control. A CCR5 specific mAb (2D7) and a CXCR4-specific mAb (12G5) were used at 0.3 mg/mL to bind to CCR5- and CXCR4-transfected cells, respectively. The data are representative of 3 experiments performed. Error bars represent SDs of 5 replicates per each data point.