CCR5 modulation on surface of CD4⁺ lymphocytes. CCR5 expression was evaluated by 2D7 Mab indirect binding, and relative surface expression was evaluated as the percentage of CCR5 expression on CD4⁺ cells. (A) CCR5 down-regulation in response to presence of 200 ng Abs for 48 hours. Total serum Abs were purified on Sepharose column and quantified by ELISA. Ig fractions were then affinity-purified on the relative CCR5 peptide and incubated with CD4⁺ lymphocytes. From the top: cells were not incubated with Igs; total Igs from healthy control; RANTES (50 nM); CCR5/peptide 3-specific Ig from LTNPs no. 22, no. 21, and no. 20. The data are representative of 2 experiments performed. (B) Kinetic of CCR5 down-regulation by 200 ng CCR5/peptide 3–specific Ig from LTNP no. 20. Total serum Abs were affinity-purified on Sepharose column and quantified by ELISA. Ig fractions were then affinity-purified on the relative CCR5 peptide and incubated with CD4⁺ lymphocytes for 1, 12, 24, and 48 hours before analysis. As positive controls, cells were not incubated with Igs, and as negative control cells were incubated with Igs from HCs. The data are representative of 3 experiments performed. (C) CCR5 expression on CD4⁺ lymphocytes from some LTNP patients carrying (no. 22, no. 20, no. 152) or not carrying (L01, M02, and C03) anti-CCR5 Abs. CD4⁺ lymphocytes from LTNPs no. 22, no. 20, and no. 152 were cultured for a further 7 days in the absence of anti-CCR5 Abs. The data are representative of 2 experiments performed. Error bars represent SDs of 3 replicates per each data point. (D) Susceptibility of infection by either HIV no. 36 (R5) or HIV no. 45 (R5, X4, R3) on CD4⁺ lymphocytes from some LTNPs carrying (no. 22, no. 20, no. 152) or not carrying (L01, M02, and C03) anti-CCR5 Abs. The data are representative of 2 experiments performed. Error bars represent SDs of 3 replicates per each data point. (E) Abs to first loop of CCR5 induce receptor endocytosis by clathrin-dependent pathway. Serum Igs from 2 LTNPs carrying CCR5 specific Abs were affinity-purified on Sepharose column and quantified by ELISA. Ig fractions were then affinity-purified on the relative CCR5 peptide (peptide 3) and incubated on CCR5-transfected cells for 48 hours in the presence or not of specific chemicals. All experiments were repeated twice, and SDs are shown.

Viremia variability correlation between patients who retained and lost anti-CCR5 Abs during follow-up period. Individual regression curves were compared between LTNP subjects changing (A) or maintaining (B) anti-CCR5 Abs. Samples were evaluated at the time of entry and at least twice during 3 years of follow-up. Median slope values per each group of LTNPs are shown. Analysis was performed by the Mann-Whitney test.

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.

Virus neutralization assays carried out with Ig fractions from 5 LTNP sera displaying anti-CCR5 Abs. A neutralizing anti-CD4 mAb (SIM4) and a neutralizing serum from a patient with AIDS (tested at the same dilutions of the LTNP sera) were used as positive control. As negative control, a pool of sera from 5 HCs was also used. The data are expressed as the percentage of infectivity reduction and are representative of 3 experiments. (A) Mediated antibody infectivity reduction obtained with subtype B isolate HIV-36. (B) Mediated antibody infectivity reduction obtained with subtype B isolate HIV-40.