Figure 7
Induction of anti-V3 Abs with phagocytic activity in vaccine recipients. The phagocytic activity of anti-V3 Abs in plasma of 7 vaccinees (4 VAX003 and 3 VAX004) was assessed using fluorophore Neutravidin™ beads coated with a biotinylated, cyclic, full-length V3 peptide (4 µg V3 peptide mixed with 2 µg scrambled peptide for 10 µL of beads/96 wells) for 2 h at 37 °C, washed, and resuspended in PBS with 0.1% BSA. Beads (9 × 105/10 µL/well) were incubated with serially diluted plasma, anti-V3 mAb, or irrelevant mAb for 2 h at 37 °C, washed, and added to THP-1 cells (2.5 × 104/well; ATCC®). After overnight incubation, phagocytosis was measured by flow cytometry. ADCP score was calculated by multiplying the percentage of bead-bearing cells with geometric mean intensity of the cells and subtracting background score. (a) Antibody-dependent cellular phagocytosis (ADCP) activities of plasma from 1 vaccinee were diluted 2-fold 6 times from 1:40. Plasma samples from early, mid-, and final time points were compared with plasma from a placebo recipient. Averages and standard error from 4 replicates tested in 2 independent experiments are shown. (b) ADCP activities were measured in plasma from 7 vaccinees collected at early, mid-, and final time points and shown as area under the curve (AUC ± standard error) of ADCP scores. Cutoff value (dotted lines) was determined based on mean plus 3 standard deviations of placebo ADCP scores. (c) Correlation between ADCP score and ELISA V3-binding Ab levels was analyzed by the Spearman test. (d) V3-specific ADCP and V3 Ab-mediated neutralization activities were correlated by the Spearman test.
