The <i>T</i>. <i>congolense</i> HpHbR is an epimastigote expressed Hb receptor.

<p>In the life cycle of <i>T</i>. <i>congolense</i>, HpHbR is expressed predominantly in the epimastigotes that inhabit the mouthparts of the tsetse fly, where it binds to Hb present in the blood meal of the fly. Here the receptor functions in the context of the major epimastigote surface protein, GARP. In the structure figure TcHpHbR is green, GARP is light blue, and Hb is orange and red.</p

The structures of trypanosome surface proteins.

<p><b>A.</b> The structures of the major surface proteins of the <i>T</i>. <i>congolense</i> epimastigote, the glutamic acid rich protein (GARP) and of the <i>T</i>. <i>brucei</i> bloodstream form, the variant surface glycoprotein (VSG), and the structures of the <i>T</i>. <i>brucei and T</i>. <i>congolense</i> haptoglobin–hemoglobin receptors (TbHpHbR and TcHpHbR). Both TbHpHbR and TbVSG are elongated by additional C-terminal domains, which are not represented [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006055#ppat.1006055.ref010" target="_blank">10</a>]. <b>B.</b> The structure of a complex of two TcHpHbR bound to a single hemoglobin tetramer. The receptors are coupled to the cell membrane by a GPI anchor and will tilt in order to simultaneously bind to a single hemoglobin. <b>C.</b> The structure of a complex of two TbHpHbR bound to a haptoglobin–hemoglobin tetramer (silver and gold), showing how the kink in the receptor allows two membrane-linked TbHpHbR to simultaneously bind to a single HpHb.</p

The <i>T. brucei</i> HpHbR is a bloodstream form HpHb receptor.

<p>In the life cycle of <i>T</i>. <i>brucei</i>, the epimastigotes inhabit the salivary glands of the tsetse fly and do not express HpHbR. Instead, TbHpHbR is predominantly expressed in the bloodstream form, in which it acts as an HpHb receptor that exists within the densely packed VSG layer. In the structure figure HpHbR is blue, VSG is blue-white, and HpHb is yellow, orange, and red. The ovals represent the C-terminal domains of the VSG and HpHbR. These lie between the N-terminal domains and the membrane, but their relative locations are uncertain.</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

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

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

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

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

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

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