Figure 3. Importance of the SD3 of PfEMP1-DBL1α in anti-rosetting response. A:

Residual activity of pIgG_IT4var60 after absorption on the SD3-loop peptide of NTS-DBL1α of IT4var60 (KVKDTCQGYNNSGYRIYCS). ELISA plates were coated with 5 µg/ml of peptide and the absorption of the pIgGs was verified by adding 10 µg/ml of the different pIgGs followed by ALP-conjugated secondary antibody. The peptide-absorbed/non-absorbed pIgGs were tested for surface reactivity by flow cytometry at 10 µg/ml and for capacity to disrupt rosettes of the homologous parasite at 250 µg/ml. As control pIgG were absorbed on the same peptide with scrambled sequence (CTSSKDYIYVQGCNNRGYK). All results are shown as relative reactivity as compared to pIgG (set to 100%). Bars show the mean of six independent experiments ± SD. B: pRBC surface reactivity of sera from rats on FCR3S1.2/IT4var60 as detected by an Alexa488-conjugated secondary antibody and visualized by flow cytometry. The rats were immunized with subdomain 1 (SD1; aa 1–119; brown), subdomain 2 (SD2; aa 120–272; light blue) or subdomain 3 (SD3; aa 273–393; dark blue) of IT4var60. Reactivity of a pre-immune rat serum is shown in red. C: Rosette disruption activity of sera of rats immunized as described under A or with full length NTS DBL1α (dilution 1∶5). Presented are the rosetting levels relative to a control incubated with PBS. Six different experiments were performed in duplicate and bars indicate SEM (Standard error of the mean). *** = p<0.001 as compared to pre-immune serum.