Polyclonal antibodies to PfEMP1 inhibit rosetting and induce phagocytosis of heterologous rosetting laboratory strains.

<p>a) Rosette inhibition assays to determine the dose-dependent effects of PfEMP1 antibodies on homologous and heterologous rosetting laboratory strains. Data are compared to a control with no added antibody, which contained at least 40% of IEs in rosettes. Mean and standard deviation of triplicate values are shown. IC50: concentration of antibody giving 50% rosette inhibition. b) Rosette inhibition assay as above with 1 mg/ml of antibody, except for the Anti-Ros pool which consisted of a mixture of 0.1 mg/ml of each antibody (to HB3var6, TM284var1, ITvar60, Muz12var1, TM180var1 and ITvar9). Controls are as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat-1002665-g004" target="_blank">Figure 4b</a>. c) Phagocytosis assay of opsonised IT/PAR+ IEs co-incubated with the monocytic cell line Thp-1 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat.1002665-Ghumra1" target="_blank">[13]</a>. Data are shown as percentage of the positive control opsonised with a rabbit anti-human erythrocyte antibody. Both homologous and heterologous antibodies induce phagocytosis of IT/PAR+ IEs. Control Rab: negative control of IgG from a non-immunized rabbit.</p

Polyclonal antibodies to PfEMP1 variants from laboratory strains show surface reactivity and rosette inhibition with <i>P. falciparum</i> clinical isolates.

<p>a) Clinical isolates were tested with PfEMP1 antibodies and controls for surface reactivity by live cell IFA (0.4 mg/ml) and rosette inhibition (1 mg/ml). The rosette frequency (RF), percentage of IgM-positive IEs (IgM+) and percentage of PfEMP1 antibody positive IEs (PfEMP1+, positive with either HB3var6 or TM284var1 antibodies) are shown for each isolate. The PfEMP1 antibodies that showed surface staining with each isolate are indicated by the shaded boxes. Positive surface staining was defined as punctate fluorescence specific to live IEs by IFA (as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat-1002665-g002" target="_blank">Figure 2</a>). The percentage rosette inhibition is shown inside each rectangle for all isolate/antibody combinations with >25% rosette inhibition. The controls are as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat-1002665-g004" target="_blank">Figure 4b</a>, and the Anti-Ros Pool is as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat-1002665-g005" target="_blank">Figure 5b</a>. The Anti-Ros pool was tested for rosette inhibition only. The dotted line separates isolates in which RF closely matches the percentage of IgM-positive IEs (above) from those in which the percentage of IgM-positive positive IEs is substantially lower than the rosette frequency (below). b) Flow cytometry of clinical isolate MAL43 with 0.4 mg/ml of total IgG from a non-immunised rabbit (negative control, left panel) and antibodies to TM284var1 (middle panel). IEs stained with Hoechst are in the right half, and antibody-positive IEs stained with Alexa Fluor 488 are in the upper right quadrant. An overlay of histograms (right panel) shows a clear population of stained IEs (blue line, second peak) distinct from the rabbit IgG control (red line, single peak). c) Five clinical isolates were tested by flow cytometry with the PfEMP1 antibody and control panel. The histograms show the negative controls, anti-PfEMP1 positive and IgM-positive IEs. The “negative PfEMP1 Ab” was antibody to TM180var1 and the IgM-negative control was a mouse IgG1 isotype control.</p

Polyclonal antibodies to PfEMP1 recognize the surface of homologous and heterologous live IEs.

<p>a) An example of the determination of the immunofluorescence end titre. Flow cytometry histograms showing the titration of antibodies to ITvar60 against IT/PAR+ parasites, compared to a non-immunized rabbit IgG control. The end titre (defined here as the lowest concentration of antibody giving surface staining above rabbit IgG background levels of more than 50% of the positive IE subpopulation) was 0.1 µg/ml. b) PfEMP1 antibodies (four-fold dilutions of total IgG starting at 400 µg/ml) were tested in IFA or flow cytometry against <i>P. falciparum</i> laboratory strains with various different adhesion phenotypes as indicated. The end titre for each antibody/parasite combination is shown inside each rectangle, with homologous antibody/parasite combinations being outlined in bold. Negative controls were non-immunized rabbit IgG control, and antibodies against NTS-DBLα from a non-rosetting Group A PfEMP1 variant (Non-ros Group A: HB3var3, expressed by HB3-HBEC which are non-rosetting parasites selected for binding to human brain endothelial cells <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat.1002665-Claessens2" target="_blank">[83]</a>). *The HB3R+ parasites contain a subpopulation of non-rosetting HB3var3-expressing IEs (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat.1002665.s007" target="_blank">Table S1</a>) that are distinct from the IgM-positive HB3var6-expressing rosetting IEs.</p

Polyclonal antibodies to PfEMP1 recognize IgM-positive IEs.

<p>HB3R+ live IEs were stained with a mixture of mouse mAb anti-human IgM (1/500 dilution) and rabbit polyclonal HB3var6 NTS-DBLα antibodies (20 µg/ml) (left column) or a mixture of mouse IgG isotype control and non-immunized rabbit IgG control (right column). Secondary incubation was with a mixture of Alexa 488 conjugated anti-rabbit IgG (1/1000) and Alexa 594-conjugated anti-mouse IgG (1/1000). IEs were stained with DAPI (1 µg/ml; scale bar 10 µm). The PfEMP1 antibodies (left column, bottom panel) stained IEs that were also positive for human IgM (left column, middle panel). Camera exposure settings were identical for PfEMP1 antibodies and controls except for human IgM/PfEMP1 with Alexa Fluor 488 which was taken at a shorter exposure setting (20 msecs) than the control (200 msecs), due to the brightness of the signal.</p

Selection for IgM yields rosetting IEs that are recognised by heterologous polyclonal PfEMP1 antibodies.

<p>a) The culture-adapted Kenyan isolate 9197 was selected three times with anti-human IgM coated Dynabeads. Comparison of the unselected and selected lines by flow cytometry showed that the IgM-selected parasites were recognised by cross-reactive PfEMP1 antibodies to HB3var6. The percentage of IEs stained with Alexa Fluor 488 are shown in the upper right quadrant. b) An IFA with dual staining (AlexaFluor 488 anti-rabbit IgG to detect PfEMP1 antibody and AlexaFluor 594 anti-mouse IgG to detect anti-human IgM) shows that the same subpopulation of IEs bound both IgM and HB3var6 antibodies. IEs were stained with DAPI (1 µg/ml; scale bar 10 µm). c) Trypsin sensitivity of surface antigens recognised by HB3var6 antibodies. Trypsinisation is as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat-1002665-g002" target="_blank">Figure 2</a>. The percentage of IEs stained with Alexa Fluor 488 are shown in the upper right quadrant.</p

Identification of key surface antigens (Group A PfEMP1 variants) of <i>P. falciparum</i> rosetting parasites and production of recombinant proteins for immunization.

<p>a) PfEMP1 domain architecture of the predominantly expressed variants from <i>P. falciparum</i> rosetting laboratory strains. The previously described rosetting variant ITvar9 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat.1002665-Ghumra1" target="_blank">[13]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat.1002665-Rowe2" target="_blank">[22]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat.1002665-Claessens1" target="_blank">[45]</a> is shown for comparison. Domain types are based on conserved motifs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat.1002665-Rask1" target="_blank">[6]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat.1002665-Smith1" target="_blank">[51]</a>. NTS: N-Terminal Segment; DBL: Duffy Binding Like; CIDR: Cysteine-rich InterDomain Region; ATS: Acidic Terminal Segment; TM: TransMembrane region. *The IT isolate was originally from Brazil, however following cross-contamination of parasite cultures in the early1980s, current IT/FCR3 strains are thought to be of South-East Asian origin <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat.1002665-Mu1" target="_blank">[88]</a>. The Genbank accession numbers for these sequences are Y13402 (<i>ITvar9/R29var1</i>), EF158099 (<i>ITvar60</i>), JQ684046 (<i>TM284var1</i>), JQ684047 (<i>TM180var1</i>) and JQ684048 (<i>Muz12var1</i>). The <i>HB3var6</i> sequence can be obtained from <a href="http://www.broadinstitute.org/annotation/%20genome/plasmodium_falciparum_spp/MultiHome.html" target="_blank">http://www.broadinstitute.org/annotation/ genome/plasmodium_falciparum_spp/MultiHome.html</a> gene reference PFHG_02274.1. b) Northern blots of RNA from isogenic rosetting (R+) and non-rosetting (R−) parasites probed with a PfEMP1 domain from the rosette-specific variant for each strain (R+ DBL probe, high stringency) and with an Exon II probe (moderate stringency), which detects all <i>var</i> genes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat.1002665-Ghumra2" target="_blank">[50]</a>. Arrows indicate the major rosette-specific <i>var</i> gene transcript in each strain. Equal loading of R+ and R− RNA was confirmed by staining with ethidium bromide (Et Br). c) Production of recombinant NTS-DBLα domains in <i>E. coli</i> to immunize rabbits. 1: TM180var1, 2: Muz12var1, 3:TM284var1, 4: ITvar60, 5:HB3var6. M: molecular weight marker; R: reduced; NR: non-reduced.</p

Polyclonal antibodies to PfEMP1 recognize the surface of homologous live infected erythrocytes (IEs).

<p>a) Live cell ImmunoFluorescence Assay (IFA) with antibodies to HB3var6 (1/50 dilution) tested on the homologous parasite (HB3R+). DAPI staining (1 µg/ml) shows the position of IEs (upper panel; scale bar 10 µm). PfEMP1 antibody is detected by highly cross-absorbed Alex Fluor 488-conjugated anti-rabbit IgG (1/500 dilution, middle and lower panels). Specific staining of IEs is seen as punctate fluorescence over the whole IE surface (middle panel, white arrows). Unstained IEs show pale smooth background fluorescence (middle panel, white arrowhead). If the plane of focus is adjusted, stained IEs show mainly rim fluorescence (lower panel). Rosettes are not seen in these images because they are disrupted by the PfEMP1 antibodies. b) IFA with antibodies from a non-immunized control rabbit (1/50 dilution) tested on HB3R+ parasite culture. Upper panel: DAPI staining shows the position of IEs (scale bar 10 µm). Lower panel: highly cross-absorbed Alex Fluor 488-conjugated anti-rabbit IgG gives no specific staining on IEs. Camera exposure settings and image handling for Alexa Fluor 488 images were identical for PfEMP1 antibody and control pictures. c) Flow cytometry of live IEs of <i>P. falciparum</i> rosetting strains stained with homologous PfEMP1 antibodies (HB3R+ parasites with HB3var6 antibodies; TM284R+ parasites with TM284var1 antibodies; IT/PAR+ parasites with ITvar60 antibodies; Muz12R+ parasites with Muz12var1 antibodies; TM180R+ parasites with TM180var1 antibodies; IT/R29 parasites with ITvar9 antibodies). Negative control rabbit IgG from a non-immunized rabbit (left column) and PfEMP1 antibodies (middle column) were tested at 100 µg/ml of total IgG. IEs were stained with Hoechst and rabbit IgG bound to the surface of erythrocytes was detected with highly cross-absorbed Alex Fluor 488-conjugated anti-rabbit IgG at 1/500 dilution. The percentage of Hoechst-stained IEs that were stained with Alexa Fluor 488 is shown in the upper right quadrant. The IE molecules recognised by PfEMP1 antibodies were sensitive to trypsin (right column) (10 µg/ml trypsin for 5 mins at room temperature (RT), followed by 1 mg/ml of trypsin inhibitor for 5 mins at RT), except for parasite strain IT/PAR+, in which the surface molecules recognized by ITvar60 antibodies were trypsin-resistant, even at 1 mg/ml of trypsin. Rabbit polyclonal antibodies to ITvar9 expressed by IT/R29 rosetting parasites have been reported previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002665#ppat.1002665-Ghumra1" target="_blank">[13]</a>, and are included here in all figures for comparison with the newly generated antibodies to the five other rosetting strains.</p