Different forms of <i>Babesia divergens</i> in human RBCs as seen on a Giemsa-stained smear from <i>in vitro</i> cultured parasites (ring, dividing figure eights, Maltese cross parasites, and multiply infected RBCs).

<p>Different forms of <i>Babesia divergens</i> in human RBCs as seen on a Giemsa-stained smear from <i>in vitro</i> cultured parasites (ring, dividing figure eights, Maltese cross parasites, and multiply infected RBCs).</p

Apical localization of PfRH5 in the merozoite.

<p>A Mature Dd2 schizonts were labeled with rabbit anti-PfRH5 IgG and counterstained with FITC-labeled anti-rabbit IgG (green) using immunofluorescence microscopy. B Colocalization studies: Mature Dd2 schizonts were double-labeled with anti-PfRH5 IgG (red) and anti-RhopH3 (green) monoclonal antibody. The merged staining (yellow) by both antibodies indicates their similar location within the parasite. C Immunoelectron microscopy confirming the localization of PfRH5 in the rhoptries within the developing merozoites in the Dd2 schizont. Parasites were fixed with 1% paraformaldehyde and 0.1% glutaraldehyde. Staining was detected with rabbit anti-RH5 and anti-rabbit gold (10 nm). Scale bar represents 500 nm.</p

Cartoon of PfRH ligands and characterization in the parasite.

<p>A. Schematic depiction of different members of the PfRH family, showing location of the signal peptide, region of homology among the various RH ligands and the trans-membrane region at the C-termini of the proteins. The bar at the bottom of PfRH5 marks the region of PfRH5 that was expressed in <i>E. coli</i>. B. Expression of a recombinant 43-kDa protein of PfRH5 (rRH5), chosen on the basis of homology with putative binding domains of <i>P. vivax</i> reticulocyte-binding protein 1 (PvRBP1) and PfRH4. Arrow indicates rRH5 after purification on GST-agarose column. C. Western Blot and immunoprecipitation analysis of asynchronous Dd2 parasite lysates with anti-rRH5 antibodies (IM). Pre-immune serum (PI) was used as a negative control. Arrow indicates specific 63 kDA RH5 protein seen in lysates.</p

PfRH5 binds to a ∼32 kDa protein on human erythrocytes.

<p>Normal erythrocyte ghost cells were separated by SDS–PAGE, and the gel was blotted and incubated with GST (the fusion partner of rRH5) or rRH5 or native parasite culture supernatant. After extensive washing, bound protein was detected by rabbit anti-RH5-GST and blots were processed by enhanced chemiluminescence (ECL, Amersham Biosciences). A specific target protein of ∼32 kDA is seen in rRH5 lane (marked with arrow) and Dd2 lanes (marked with arrow). Asterisk denotes a non-specific protein band that appears in control GST and other lanes. Parallel blots were probed with anti-GPA/B or anti GPC/D antibodies to define positions of the glycophorins relative to the 32 kDA band. Pre-immune rabbit sera did not yield any reactivity (PI).</p

Erythrocyte-binding activity of native PfRH5.

<p>A Binding of the native PfRH5 protein in the HB3 culture supernatant incubated with untreated (WT) erythrocytes, variously enzyme-treated erythrocytes (N: neuraminidase; HT, high trypsin; C, chymotrypsin). The PfRH5 parasite protein was detected in the eluate fractions by immunoprecipitation with anti-RH5 antibodies in WT, N and C lanes but not HT. B The same eluate samples from (A) were used for the detection of EBA-175 by immunoprecipitation. EBA-175 binds to wild type and chymotrypsin-treated erythrocytes but not to neuraminidase and high trypsin-treated erythrocytes C Binding of the native RH5 protein to erythrocytes treated with lower concentrations of trypsin (LT low trypsin and MT: moderate trypsin). D The eluate samples from (C) were used for the detection of EBA-175 by immunoprecipitation. EBA-175 binds to untreated erythrocytes but not to low-trypsin- and moderate-trypsin-treated erythrocytes.</p

Erythrocyte-binding activity of rRH5.

<p>A 0.6 µmoles of rRH5 were used in binding assays with untreated RBCs and RBCs treated with neuraminidase- (N), low-trypsin- (LT), medium-trypsin- (MT), high-trypsin- (HT) and chymotrypsin, followed by elution and immunoprecipitation with anti-RH5 antibodies. Gels containing the immuno-precipitates were blotted and probed with anti-RH5 antibodies. B Antibodies to rRH5 block the erythrocyte binding of the recombinant protein. Total IgG from rabbits immunized with rRH5, blocks erythrocyte binding of the RH5 recombinant protein. 0.6 µmoles of rRH5 was incubated with normal erythrocytes in the presence of purified IgG from rabbit sera at final concentrations of 0–10 µg/100 µl.</p

Enzymatic (neuraminidase) treatment of erythrocytes eliminates the inhibitory effects of anti-EBA-175_RII antibodies but has only a mild effect on anti-rRH5 sera.

<p>Invasion inhibition assays with 3D7 and neuraminidase-treated cells (100 µg/mL antibody used) show that any inhibition due to the presence of anti-EBA-175_RII antibodies is masked by the removal of ligands with sialic acid compared to untreated cells (<b>A</b>; anti-EBA-175_RII and IgG shown as solid black and solid white bars, respectively). Anti-rRH5 alone (<b>B</b>; solid blue bars), or in combination (<b>C</b>; anti-EBA-175_RII/rRH5 and anti-EBA-175_RII+anti-rRH5 shown as solid red and solid green bars, respectively) is still able to significantly inhibit growth in treated cells as a sialic acid independent pathway is utilized by the RH5 antigen.</p

Recombinant EBA-175 and RH5 antigens are stable, pure and expressed in the correct conformation.

<p>The non-reduced (Lane 1 of panel <b>A</b>) and the reduced elution (Lane 2 of panel <b>A</b>; both visualized by Coomassie staining) of region II of EBA-175 synthesized using the yeast expression system <i>Pichia pastoris</i>, and the binding of this recombinant to normal erythrocytes (panel <b>B</b>), confirm correct expression and conformation of the EBA-175_RII antigen with the expected product of ∼80 kDa (indicated by the arrows in Lane 2 of panel <b>A</b> and panel <b>B</b>). The elution of full length RH5 synthesized using the wheat-germ synthesis (panel <b>C</b>, indicated by the arrow), and binding of rRH5 (panel <b>D</b>) to normal erythrocytes indicates functional conformity of this recombinant antigen, as shown by the presence of a single product at the expected size of ∼63 kDa (indicated by arrows in both <b>C</b> and <b>D</b>).</p

Independent Antibody Interaction and Co-Operative Antibody Interaction Models.

<p><b><i>Independent Antibody Interaction Model</i></b> (Panel <b>A</b>): If the ligand-receptor interactions are independent of each other, invasion by either EBA-175 (Route 1) or by RH5 (Route 2) is not affected by the other. Thus, in the presence of both anti-EBA-175_RII and anti-rRH5 antibodies, the expected inhibition from the combination anti-sera is ADDITIVE (synergistic) when compared to the inhibition from the individual anti-sera. <b><i>Co-Operative Antibody Interaction Model</i></b> (Panel <b>B</b>): If the ligand-receptors act in a co-operative method, then invasion by EBA-175 (Route 1) and RH5 (Route 2) are not independent of each other. Thus, in the presence of anti-EBA-175_RII and anti-rRH5 antibodies, the expected inhibition from the combination anti-sera is only as effective as the most active individual antibody. <b><i>Antibody Steric Hindrance</i></b> (Panels <b>C</b> and <b>D</b>): Data suggests that EBA-175 abundance is greater than RH5 and is possibly released before RH5 (Ord et al, unpublished observations). At LOW antibody concentrations (<b>C</b>), there is no possible hindrance of RH5 by EBA-175, and all available antibodies are able to bind to their respective ligands independently. This is observed as growth inhibition with the combination anti-sera being more effective than that observed with the individual antibodies, i.e., it follows the Independent Antibody Interaction Model (<b>A</b>). Conversely, at HIGH antibody concentrations (<b>D</b>), anti-EBA-175 antibodies are able to bind to available EBA-175 ligands but they sterically hinder some RH5 antibody/ligand interactions, leaving some RH5 ligands available for invasion through the RH5 ligand/receptor pathways. This is observed as growth inhibition with the combination anti-sera being only as effective as anti-EBA-175_RII sera alone, i.e. it follows the Co-Operative Antibody Interaction Model (<b>B</b>).</p

Anti-sera against the hybrid vaccine show synergistic effects at low concentrations.

<p>The invasion inhibition of both combination sera are greater than those obtained from the individual sera at the lowest concentrations used, 1 µg/mL. The combinations contain 50% of each individual immunogen (in the case of the combination vaccination, anti-EBA-175_RII/rRH5) or sera (in the case of the in-tube combination, anti-EBA-175_RII+anti-rRH5). However, by 100 µg/mL, the synergistic effects of the combinations are no longer apparent, and the inhibition from the combinations is equivalent to ∼50% contribution from the two individual sera (anti-EBA-175_RII shown by black bars, anti-rRH5 shown by blue bars, anti-EBA-175_RII/rRH5 shown by red bars, anti-EBA-175_RII+anti-rRH5 shown by green, control IgG shown by white bars).</p