Total and IgG subclass reactivity to PvRBPs in young Papua New Guinean children.

<p>Total and IgG subclass reactivity to PvRBPs in young Papua New Guinean children.</p

Erythrocyte-binding preferences of 6 PvRBPs.

<p>A: Bar charts showing the percentage of binding of CD71-PECy5 (control), PvRBP1a, PvRBP1b, PvRBP2a, PvRBP2b, PvRBP2cNB, PvRBP2-P2 to mature erythrocytes (not stained with thiazole orange, TO-) vs reticulocytes (stained with thiazole orange, TO+) populations. Error bars represent SEM of 7 or 9 independent repeats. B: Dot plots show the binding of six PvRBP proteins to an enriched reticulocyte population. Binding was detected using an anti-PvRBP rabbit IgG antibody followed by a secondary anti-rabbit Alexa 647 antibody.</p

Detection of PvRBPs by rabbit anti-PvRBP polyclonal antibodies by ELISA.

<p>Microtiter wells were coated with each PvRBP per well as shown by symbols on the left. Solid lines show specific anti-PvRBPs polyclonal antibodies (top label) added to each plate in a dilution series. The optical density (OD) was measured at 405 nm. Mean OD values from duplicated wells and standard deviation are shown.</p

Association between total IgG to 6 PvRBPs age and exposure in 224 young Papua New Guinea children.

<p>A & B: Data are plotted as Lowess smoothed curves of total IgG levels (arbitrary units) by age and life-time exposure respectively. Life-time exposure was calculated as age multiplied by molFOB. Correlation coefficients (rho) and P-values from Spearman’s rank test. <b>C:</b> Boxplots show median total IgG levels (arbitrary units) and range (whiskers) by infection status detected by PCR. Clear boxes show low antibody levels, dotted boxes show medium antibody levels and grey boxes show high antibody levels. P-values from Kruskal-Wallis test. For all analysis, P < 0.05 were deemed significant.</p

Association between total and IgG subclasses to 6 PvRBPs and protection against clinical malaria (density > 500/μL) in 224 young Papua New Guinean children.

<p>Data are plotted as exposure (molFOB), age, season and village of residency adjusted incidence rate ratios and 95% confidence intervals. Incidence rate ratios, 95% confidence intervals and P-values from GEE models. P < 0.05 were deemed significant. ****P < 0.001; ***P = 0.001; ** P > 0.001 to 0.01; *P > 0.01 to 0.05.</p

None of CR1 15–25 variants interact with <i>P. falciparum</i> protein Rh4.

<p>(<b>A</b>) In an SPR experiment, no CR1 15–25 variant had significant affinity for immobilized Rh4.9, unlike positive controls sCR1 and CR1 1–3 (26). Other constructs lacking CCPs 1–3 (CR1 10–11, CR1 15–17, CR1 20–23, CR1 21–22 and CR1 24–25(KR)) were likewise lacking in affinity for Rh4.9 and, like BSA, served as negative controls. (<b>B</b>) The PfRh4 invasion pathway was not inhibited in the presence of CR1 15–25 variants. Parasite strains 3D7 (black bars for untreated, white bars for <i>nm</i>-treated erythrocytes) and W2mefΔRh4 (grey bars, untreated erythrocytes) were tested in growth assays in the presence of final concentrations of 0.5 mg/ml of the four CR1 15–25 variants (KR, ER <i>etc.</i>). Growth (% of control) on the y-axis refers to the % parasitemia in the presence of CR1 constructs relative to the % parasitemia with the addition of PBS (arbitrarily set to be 100%).</p

Recombinant CR1 truncations produced for the current study.

<p>The Coomassie-stained polyacrylamide gels show the results of electrophoresis of successive fractions (with increasing elution volumes, <i>V</i>_e) from size-exclusion chromatography for CR1-21, CR1 21–22, CR1 20–23 and CR1 15–25 (the K1590,R1601 variant); reducing (R) conditions in some lanes would reveal products of intra-modular proteolytic “clipping” that would be held together by disulfide bonds under non-reducing (NR) conditions.</p

Affinities for C1q.

<p>(<b>A</b>) Representative SPR-derived binding curves for CR1 15–25 variants flowed over a CM5 chip loaded (via amine coupling) with C1q (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034820#pone.0034820.s004" target="_blank">Fig. S4</a>). <i>Upper:</i> sensorgrams recorded for a range of CR1 15–25 concentrations (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034820#pone-0034820-g003" target="_blank">Fig. 3</a>). <i>Lower:</i> plots of response versus [CR1 15–25] used to calculate (see Methods) the <i>K</i>_D values listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034820#pone-0034820-t002" target="_blank">Table 2</a>. CR1 variants that are widespread amongst Caucasoids (KR) or Africans (EG) bind equally to C1q. (<b>B</b>) Data obtained by ELISA showing that C1q (140 ng.mL⁻¹, average of three experiments) bound equally to each of the four variants of CR1 15–25 (adhered to the micro-titer plate). In these experiments, sCR1 was used as a positive control. Error bars represent standard errors of the mean.</p

CR1 15–25 variants do not differ in ability to disrupt <i>P. falciparum</i> rosetting.

<p>(<b>A</b>) Both CR1 10–11 (identical to CR1 17–18) and single-module CR1 17 can disrupt rosetting of <i>P. falciparum</i> clone IT/R29, as can anti-CR1 antibody (J3B11) and CR1 15–17 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034820#pone.0034820-Rowe3" target="_blank">[27]</a> used here as positive controls. The truncation mutants corresponding to CCPs 20–23 (<b>B</b>) and the CR1 homologue, factor H (FH, full-length or fragments as indicated below) (<b>C</b>) did not disrupt rosetting, and served as negative controls. (<b>D</b>) A comparison of the four CR1 15–25 variants suggested they were all equally as effective as full-length sCR1 and CR1 15–17 in terms of rosette disruption. KP: 50 mM potassium phosphate buffer; The mean and standard error of at least four experiments for each graph are shown. *** p<0.001 by ANOVA and Tukey's multiple comparison test.</p

AUC data for four CR1 15–25 variants.

1<p>Symbols in this table: <i>s</i> = sedimentation coefficient (in Svedbergs); <i>f/f_o</i> = frictional ratio from SEDFIT and from the combination of s and molecular mass (M); <i>R_h</i> = hydrodynamic radius; KR, <i>etc.</i> refer to CR1 15–25 (KR), <i>etc.</i></p