Novel antimalarial antibodies highlight the importance of the antibody Fc region in mediating protection by Richard J. Pleass, Solabomi A. Ogun, David H. McGuinness, Jan G. J. van de Winkel, Anthony A. Holder, and Jenny M. Woof Blood Volume 102(13):4424-4430 December 15, 2003 ©2003 by American Society of Hematology

SDS-PAGE analysis of purified Abs and diabody. SDS-PAGE analysis of purified Abs and diabody. (A) Purified anti-MSP119 mouse IgG2b (lanes 1, 4, 7, and 10), human IgG1 (lanes 2, 5, 8, and 11), and human IgA1 (lanes 3, 6, 9, and 12) were subjected to SDS-PAGE under nonreducing (lanes 1-3 and 7-8) or reducing (lanes 4-5 and 10-12) conditions on 8.2% polyacrylamide gels, and stained with Coomassie blue (lanes 1-6) or immunoblotted (lanes 7-12). (B) The Abs detect recombinant GST-MSP119 fusion protein (lanes 1-3) or a fragment of MSP1 in an extract of P yoelii YM merozoites electrophoresed under nonreducing conditions (lanes 4-6) and transferred to nitrocellulose. Detecting Abs: lanes 1 and 4, mouse IgG2b; lanes 2 and 5, human IgG1; and lanes 3 and 6, human IgA1. (C) Purified FcγRI × MSP119 diabody was run under nonreducing (lanes 1 and 3) or reducing (lanes 2 and 4) conditions. Samples were either blotted and probed with anti-His (lanes 1 and 2) or stained with Coomassie blue (lanes 3 and 4). The diabody detected a blot of recombinant GST-MSP119 fusion protein run under nonreducing conditions (lane 6). Secondary anti-His Ab did not cross-react with the fusion protein alone (lane 5). Richard J. Pleass et al. Blood 2003;102:4424-4430 ©2003 by American Society of Hematology

Immunofluorescence and SPR analysis. Immunofluorescence and SPR analysis. (A) Immunofluorescence patterns of Abs or diabodies reactive with MSP119 on methanol-acetone-fixed smears of merozoites and erythrocytes infected with P yoelii YM. The smears were incubated with human IgG1 (iii,iv), human IgA1 (v,vi) and the FcγRI × MSP119 diabody (ii). No specific fluorescence was detected with an irrelevant FITC-labeled Ab (i) or with the secondary F(ab′)2 anti-Ig Ab alone (not shown). Original magnification: panels i-iii, v, × 40; iv, vi, × 100. (B) SPR association and dissociation curves of Ab binding to MSP119 immobilized on a CM5 sensor chip. Abs were injected into flow at time 0, and replaced with buffer at the points indicated by vertical arrows. Richard J. Pleass et al. Blood 2003;102:4424-4430 ©2003 by American Society of Hematology

Stimulation of neutrophil NADPH oxidative bursts using Abs attached to GST-MSP119-coated microtiter plates. Stimulation of neutrophil NADPH oxidative bursts using Abs attached to GST-MSP119-coated microtiter plates. Chemiluminescence (CL; arbitary units) was induced by (A) anti-MSP119 human IgA, or (B) antihuman MSP119 human IgG1 at (▪) 1 × 10-6 M, (▴) 5 × 10-7 M, (•) 1 × 10-7 M, and (□) 5 × 10-8 M. Isotype controls (○) IgA1-NIP or IgG1-NIP, or (♦) MSP119 specific mouse IgG2b at 1 × 10-6 M. (C) Diabody or Ab at 1 × 10-6 M; (▪), FcγRI × MSP119; (•), IgG1-MSP119; (♦), IgG2b-MSP119; (□), IgG1 isotype control; ▵, no Ab or diabody. In each case the experiments were performed 4 times with neutrophils from different donors with similar results. Richard J. Pleass et al. Blood 2003;102:4424-4430 ©2003 by American Society of Hematology

Binding of merozoites to neutrophils. Binding of merozoites to neutrophils. (A) Forward and side scatter flow cytometry analysis of merozoites, neutrophils, or a mixture showing gating of different CD16+ neutrophil populations. (B) Binding of merozoites opsonized with FITC-labeled MSP119-specific human IgG1, human IgA1, or mouse IgG2b (red traces), to total neutrophils (R1) or neutrophils in gate 2 (R2). Binding of merozoites opsonized with an isotype-matched control Ab shown by the black trace. (C) Mean fluorescence intensities of opsonized merozoites binding to R1-(□) or R2-gated neutrophils (▪). Error bars indicate standard deviation from the mean. Richard J. Pleass et al. Blood 2003;102:4424-4430 ©2003 by American Society of Hematology

Neutrophil phagocytosis of merozoites. Neutrophil phagocytosis of merozoites. Confocal fluorescence microscopy of phagocytosis of labeled merozoites opsonized with FITC-labeled MSP119-specific Abs by human neutrophils. Labelled merozoites are arrowed. For percentage phagocytosis, the mean and standard deviations of 2 experiments are shown. Original magnification, × 100. Richard J. Pleass et al. Blood 2003;102:4424-4430 ©2003 by American Society of Hematology

Course of P yoelii YM parasite infection in mice. Course of P yoelii YM parasite infection in mice. Groups of 5 BALB/c mice injected with mouse IgG2b anti-MSP119 (▴), human IgG1anti-MSP119 (□), human IgA1 anti-MSP119 (○), human IgG1anti-NIP (▪), human IgA1 anti-NIP (•), FcγRI × MSP119 diabody (♦), and PBS ▵. Each point represents the geometric mean parasitemia of mice in each group at the time after parasite challenge. All mice in the mouse IgG2b group survived for 6 months; all the mice in the other groups with high parasitemia were killed on either day 4 or day 5. Richard J. Pleass et al. Blood 2003;102:4424-4430 ©2003 by American Society of Hematology