FIG. 3.

Flow cytometry analysis of the specificities and biological activities of anti-CCR5 total Igs or IgG- or IgA-enriched fractions from antisera elicited by the recombinant immunogen FHV-I2. (A) Binding of either IgA or IgG fractions (20 ng/assay) to CCR5 receptors on mouse CD4⁺ lymphocytes. Positive controls were lymphocytes treated with an anti-mouse CCR5 MAb (M20), and negative controls were lymphocytes treated only with the secondary Ab (RAM-FITC). (B) Kinetics of CCR5 down-regulation on mouse CD4⁺ lymphocytes of untreated mice by total Ig fractions (20 ng/assay) purified from FHV-I2 antisera. Lymphocytes were incubated with purified Igs for 12 and 48 h before flow analysis. Positive and negative controls were as in panel A. (C) CCR5 down-regulation obtained by incubating mouse CD4⁺ lymphocytes with purified Igs (20 ng/assay) for 48 h before flow analysis. As negative controls, target cells were incubated either with Igs purified from NMS or with only the secondary Ab (RAM-FITC). (D) Dose-response curves of CCR5 down-regulation obtained by incubating mouse CD4⁺ lymphocytes for 48 h with the indicated concentrations of Igs purified from pools of antisera obtained by either systemic or mucosal immunizations. As negative controls, mouse CD4⁺ lymphocytes were also incubated with Igs purified from FHV-WT antisera or with only the secondary Ab (RAM-FITC). These data were obtained with serum samples drawn 1 week after the third immunization.