Anti-RSP2 antibodies induce phagocytosis of RSP2-tagged UEs and complement activation. (A) Flow cytometry analysis showing that the percentage of RSP2-tagged erythrocytes labeled with mAb B4 depends on parasite load. Dilutions of 0.5% to 8% trophozoites were used and, after reinvasion, the percentages of RSP2-tagged UEs and RSP2-tagged IEs (±SD) were determined by staining with ethidium bromide. (B) Mobility of RSP2 on the surface of a UE. The close-up shows the region of interest (ROI 1) before, just after, and 1420 seconds after photobleaching. The graph shows the kinetics of fluorescence recovery (FRAP) (ROI 1) after correction for its natural decrease over time (ROI 2 and ROI 3). (C) Flow cytometry revealing RSP2-tagged erythrocytes in D10ΔRAP1, D10, and FCR3^CSA culture, using the mAb B4, the sera from anemic patients 2, 3, and 4 and the pool of sera from immune asymptomatic African donors (PIAG). An IgG2a mAb and serum 1 were used as negative controls. (D) Comparison of the opsonization capacity of B4, the sera used in panel A and the negative controls. The phagocytosis index is the number of IEs and UEs undergoing phagocytosis per 100 monocytes counted (± SD). (E) Cultured FCR3^CD36 labeled with ethidium bromide (FL2) and with mAb B4 (FL1 RSP2) or with a human anti-C3 antibody by incubation with mAb B4 and C3⁺ human serum (FL1 C3). IgG2a was used as an isotypic control (gray) for RSP2, and C3 immunostaining was used to assess the specific association of C3 with cytophilic antibodies such as mAb B4 (IgG2a).