The density of knobs on the surface of erythrocytes infected by isolate GH18 after prolonged culture <i>in vitro</i>.

<p>The relationship between time since invasion and IE surface knob density after <i>in vitro</i> culture of isolate GH18 for 8 weeks (A) or 12 weeks (B). All time/density data points <36 h as well as the common regression line (with 95% confidence interval) for the GH18 (note that the <i>ex vivo</i> data points shown in Fig. 2M are included) (C). Individual data points, as well as the linear regression line (with 95% confidence limits) for data points <36 h are shown. Adjusted regression lines (assuming parallelism) after different times of <i>in vitro</i> culture of isolate GH18 (D).</p

The density of knobs on the surface of erythrocytes infected by long-term in vitro isolates of <i>P. falciparum</i> expressing the PfEMP1 protein VAR2CSA.

<p>The relationship between time since invasion (h) and IE surface knob density (µm^–2) on erythrocytes infected by 10 genotypically distinct isolates of <i>P. falciparum</i> (isolate name in brackets), maintained in long-time <i>in vitro</i> culture and selected for expression of the PfEMP1 protein VAR2CSA by regular panning for IE adhesion to CSA (A-J). Individual data points, as well as the linear regression line (with 95% confidence limits) for data points <36 h are shown for each isolate.</p

Identification and characterization of the α₂M-binding domain in HB3VAR06.

<p>(A) Schematic representation of the domain structure of HB3VAR06. Domain nomenclature as described by Rask <i>et al</i>. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005022#ppat.1005022.ref037" target="_blank">37</a>], as well as the first and last amino acid in each domain are indicated. (B) Binding of α₂M to recombinant HB3VAR06 single-, double-, and triple-domain constructs (labeled as in panel A) as well as to full-length HB3VAR06 (FV6) measured by ELISA. Means and SD are indicated. (C) Inhibition of α₂M binding to HB3VAR06⁺ IEs by anti-sera raised against the N-terminal head structure (D1–D3), DBLξ2 (D8), and full-length HB3VAR06 (FV6), respectively, measured by flow cytometry. Means and SD relative to binding without anti-serum are indicated. (D) Simultaneous labeling of HB3VAR06⁺ IEs by α₂M and IgM (left), measured by flow cytometry. A control experiment with all detecting antibodies present but without α₂M and IgM is shown to the right. (E) Inhibition of IgM binding to HB3VAR06⁺ IEs by increasing concentrations of α₂M, measured by flow cytometry. Means and SD relative to binding in the absence of α₂M are indicated. (F) Inhibition of α₂M binding to HB3VAR06⁺ IEs by increasing concentrations of IgM, measured by flow cytometry. Means and SD relative to binding in the absence of IgM are indicated.</p

Parasite developmental age and infected erythrocyte knob density.

<p>Example atomic force micrographs of erythrocytes infected by a early schizont parasite <i>ex vivo</i> (A) or by a younger VAR2CSA-expressing trophozoite (B). Black scale bars: 1 µm. The inserts show light micrographs of the same infected erythrocytes stained by Giemsa. Close-up micrographs of knobs (C) and of a single knob (D). Black scale bars: 200 nm (C), 75 nm (D).</p

Identification of α₂M as the soluble serum factor binding HB3VAR06.

<p>(A) 2D gel electrophoresis of serum components pulled down with recombinant full-length HB3VAR06 (FV6; left) or PFD1235w-DBLβ3_D5 (right). Spots subsequently identified as α₂M and IgM in the left panel are boxed. Molecular weight (kDa) markers are shown along the left margins. (B) Binding of α₂M to recombinant full-length HB3VAR06 (FV6; left) and IT4VAR04 (FV2; right), measured by ELISA. Means and SD are indicated. (C) Binding of α₂M to HB3VAR06⁺ IEs (left) and IT4VAR04⁺ IEs (right), measured by flow cytometry. Control sample labeling (no α₂M added) is indicated by gray shading. (D) Fluorescence micrographs of DAPI-labeled HB3VAR06⁺ IEs in the presence (top) and absence (bottom) of fluorescein isothiocyanate-labeled α₂M at low (scale bar: 20 μm; left) and high (scale bar: 5 μm; right) magnification are shown.</p

Binding of native and MA-activated α₂M to HB3VAR06.

<p>(A) Titration of binding of native α₂M (black circles) and α₂M-MA (white circles) to recombinant full-length HB3VAR06 measured by ELISA. Means and SD are indicated. (B) Titration of binding of native α₂M (black circles) and α₂M-MA (white circles) to HB3VAR06⁺ IEs measured by flow cytometry. Means and SD are indicated. (C) Activation of α₂M measured by SDS gel electrophoresis of soluble and immobilized α₂M in the presence of mPEG: soluble α₂M alone (lane 1), soluble α₂M and MA (lane 2), soluble α₂M and FV6 (lane 3), bead-immobilized α₂M-FV6 complexes alone (lane 4), and bead-immobilized α₂M-FV6 complexes and MA (lane 5). While native α₂M was detectable in all lanes, activated α₂M having a higher molecular weight than native α₂M due to incorporation of mPEG was only detected in the presence of MA (lanes 2 and 5).</p

Merozoite-specific IgG according to clinical category.

<p>Levels (AU) of IgG specific for PfRH5 (A), CyRPA (B), Pf113 (C), EBA175 (D), GLURP-R0 (E) and GLURP-R2 (F) in plasma of individual children according to clinical category: SM (severe P. falciparum malaria), UM (uncomplicated P. falciparum malaria), FC (non-parasitemic febrile controls), AC (asymptomatic controls), HC (non-parasitemic healthy controls). Please refer to Materials and Methods for category definitions. The number of individuals with IgG above cut-off and the total number of individuals in each clinical category are given along the top of each panel. Horizontal lines along the top of the panels indicate statistically significant (P<0.05) differences between groups. Data presentation otherwise as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198371#pone.0198371.g001" target="_blank">Fig 1B</a>. The presented data is from one experiment.</p

Correlations (Spearman’s ρ [associated P-values] of IgG responses.

<p>Correlations (Spearman’s ρ [associated P-values] of IgG responses.</p