Rosette disruption assays with ITvar9 antisera against six <i>P. falciparum</i> rosetting laboratory strains.

<p>Antisera raised to ITvar9 domains, and paired pre-immune sera, were used at 1/20 dilution in rosette disruption assays with R29, PAR+, Muz12, HB3R+, TM284 and TM180. Antisera were as follows: A) NTS-DBL1α, B) DBL1α, C) NTS-DBL1α-CIDR1γ, D) DBL2γ, E) DBL3ε, F) DBL4δ and G) CIDR2β. Disruption of rosetting was only seen with R29 parasites. Data shown are the mean and standard deviation from three independent experiments. The control (with binding medium only added) had more than 50% of infected erythrocytes in rosettes.</p

Effectiveness of ITvar9 antibodies in various assays^#.

#<p>50% ELISA titres for the antibodies (defined as the titre giving 50% of the maximum OD) were as follows: NTS-DBL1α 1/500,000; DBL1α 1/250,000; NTS-DBL1α-CIDR1γ 1/300,000; DBL2γ 1/50,000; DBL3ε 1/200,000; DBL4δ 1/40,000 and CIDR2β 1/200,000.</p>$<p>The lowest concentration at which >50% of the infected erythrocytes in the culture showed punctate fluorescence by IFA. Values shown are µg/ml of purified total IgG.</p><p>*50% inhibitory concentration (IC50) for rosette inhibition. Values shown are µg/ml of purified total IgG.</p>†<p>The most effective antibodies in each assay are shown in bold. Values shown are µg/ml of purified total IgG.</p

Phagocytosis of R29 infected erythrocytes after opsonization with anti-PfEMP1 antibodies.

<p>Ethidium bromide stained R29-infected erythrocytes were opsonized with antibodies and incubated with the monocytic Thp-1 cell line. The percentage of Thp-1 cells that had phagocytosed one or more infected erythrocytes was assessed by flow cytometry. The positive control was 90 µg/ml rabbit-anti human erythrocyte polyclonal antibody and the negative control was media alone (no serum control). All antibodies to PfEMP1 domains were used at four different concentrations: 100 µg/ml (A), 25 µg/ml (B), 6.25 µg/ml (C) or 1.56 µg/ml (D). Antibodies directed against ITvar9 PfEMP1 domains (first seven bars of each graph) promoted phagocytosis of R29 infected erythrocytes, whereas antibodies to the NTS-DBL1α domains of other PfEMP1 variants (control PAR+, TM284, TM180 and HB3R+) did not. The effect of ITvar9 PfEMP1 antibodies was concentration-dependent, with anti-NTS-DBL1α being the most effective at low concentration (D). Values shown are means and standard deviation from duplicates.</p

Immunofluorescence assay showing that ITvar9 antibodies recognize PfEMP1 on the surface of live infected erythrocytes.

<p>R29 mature infected erythrocytes (pigmented trophozoites and schizonts) were grown to 5% parasitaemia and incubated with rabbit antisera against recombinant ITvar9 DBL and CIDR domains at 1/50 dilution. After washing, the cells were incubated with Alexa Fluor 488-labelled goat anti-rabbit IgG (Invitrogen) at 1/1000 dilution. The example shown here is the binding of anti-DBL2γ antisera, however all antisera to ITvar9 gave similar results. Punctate staining of the membrane of infected erythrocytes (green) was seen with the specific antisera (“immune”) but not with the pre-immune sera. The location of infected erythrocytes is shown by DAPI staining of the parasite (blue). Slides were viewed with a 100× objective using a Leica DM 2000 fluorescent microscope.</p

ELISA to detect binding of PfEMP1 antibodies to recombinant NTS-DBL1α.

<p>Antibodies were added to wells coated with 2 µg/ml of recombinant NTS-DBL1α protein and binding was detected using HRP-conjugated anti-rabbit IgG at 1/10,000 dilution. Antisera raised to recombinant proteins containing DBL1α (i.e. anti-NTS-DBL1α, anti-DBL1α and anti-NTS-DBL1α-CIDR1γ) all recognize the recombinant protein as expected. Antisera to DBL2γ, DBL3ε and CIDR2β do not cross-react with recombinant NTS-DBL1α. However, the antiserum to DBL4δ does shows binding to the recombinant NTS-DBL1α, suggesting that there is cross-reactivity between these two domains.</p

Rosette inhibition of antibodies depleted by absorption against NTS-DBL1α.

<p>Immunoblotting (A) and rosette inhibition (B) by pairs of antibodies that were either non-absorbed, or absorbed on NTS-DBL1α recombinant protein coupled to sepharose. A) Recombinant NTS-DBL1α protein was spotted onto nitrocellulose membrane at doubling dilutions, starting from 2 µg/ml, and incubated with 1/1000 dilution of absorbed or non-absorbed antibody. 1) non-absorbed anti-NTS-DBL1α, 2) absorbed anti-NTS-DBL1α, 3) non-absorbed anti-NTS-DBL1α-CIDR1γ, 4) absorbed anti-NTS-DBL1α-CIDR1γ, 5) non-absorbed anti-DBL3ε, 6) absorbed anti-DBL3ε, 7) non-absorbed anti-DBL4δ and 8) absorbed anti-DBL4δ. Non-absorbed antibodies to DBLα (lanes 1 and 3) and DBL4δ (lane7) recognized NTS-DBL1α recombinant protein. After absorption, however, this activity was lost (lanes 2, 4 and 8). Antibodies to DBL3ε did not recognize NTS-DBL1α recombinant protein (lanes 4 and 5). B) Rosette inhibition assays showed that the anti-rosetting activity of NTS-DBL1α antibodies was lost after absorption. Antibodies to DBL3ε and DBL4δ retained rosette-inhibitory activity after absorption, showing that their anti-rosetting effects are likely to be independent of DBL1α. Antibodies to NTS-DBL1α-CIDR1γ also retained inhibitory effects after absorption on NTS-DBL1α protein, suggesting that antibodies to the CIDR1γ domain of ITvar9 also have anti-rosetting effects. Data shown are the mean and standard deviation of triplicate determinations of rosette frequency after overnight incubation with absorbed or non-absorbed antibody diluted 1/10 from the 1 mg/ml stock used for absorption. The control (with binding medium only added) had more than 50% of infected erythrocytes in rosettes.</p

SDS-PAGE showing recombinant DBL and CIDR domains from ITvar9 expressed in <i>E. coli</i>.

<p>The purity and quality of the recombinant DBL and CIDR domains were assessed by electrophoresis of reduced and non-reduced pairs of proteins on 10% SDS-polyacrylamide gels. Two µg of protein was used per well and lanes were as follows: 1) NTS-DBL1α, 2) DBL1α, 3) DBL2γ 4) DBL3ε, 5) DBL4δ, 6) CIDR2β and 7) NTS-DBL1α-CIDR1γ. M, molecular weight marker; NR, non-reduced; R, reduced.</p

Rosette inhibition assays with ITvar9 antisera against six <i>P. falciparum</i> rosetting laboratory strains.

<p>Total IgG was used at a concentration of 100 µg/ml in rosette inhibition assays with R29, PAR+, Muz12, HB3R+, TM284 and TM180. Antisera were as follows: A) NTS-DBL1α, B) DBL1α, C) NTS-DBL1α-CIDR1γ, D) DBL2γ, E) DBL3ε, F) DBL4δ and G) CIDR2β. Inhibition of rosetting was only seen with R29 parasites. Data shown are the mean and standard deviation of triplicate determinations of rosette frequency within a single experiment. The control (with binding medium only added) had more than 50% of infected erythrocytes in rosettes.</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 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