Targeting Sialic Acid Dependent and Independent Pathways of Invasion in Plasmodium falciparum

The pathology of malaria is a consequence of the parasitaemia which develops through the cyclical asexual replication of parasites in a patient's red blood cells. Multiple parasite ligand-erythrocyte receptor interactions must occur for successful Plasmodium invasion of the human red cell. Two major malaria ligand families have been implicated in these variable ligand-receptor interactions used by Plasmodium falciparum to invade human red cells: the micronemal proteins from the Erythrocyte Binding Ligands (EBL) family and the rhoptry proteins from the Reticulocyte binding Homolog (PfRH) family. Ligands from the EBL family largely govern the sialic acid (SA) dependent pathways of invasion and the RH family ligands (except for RH1) mediate SA independent invasion. In an attempt to dissect out the invasion inhibitory effects of antibodies against ligands from both pathways, we have used EBA-175 and RH5 as model members of each pathway. Mice were immunized with either region II of EBA-175 produced in Pichia pastoris or full-length RH5 produced by the wheat germ cell-free system, or a combination of the two antigens to look for synergistic inhibitory effects of the induced antibodies. Sera obtained from these immunizations were tested for native antigen recognition and for efficacy in invasion inhibition assays. Results obtained show promise for the potential use of such hybrid vaccines to induce antibodies that can block multiple parasite ligand-red cell receptor interactions and thus inhibit parasite invasion

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

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

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

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

Dd2 parasites are not wholly dependent on the EBA-175/GPA pathway of invasion.

<p>In the presence of anti-EBA-175_RII antibodies (<b>A</b>; black bars), inhibition when using the Dd2 strain is only 60% (Ab concentration of100 µg/mL), compared to 55% inhibition from 3D7, suggesting alternative SA dependent pathways are utilized by Dd2, such as EBL-1/GPB or EBA-140/GPC (for <b>A</b>, <b>B</b> and <b>C</b>, Dd2 with its control IgG shown as solid white bars). Although anti-rRH5 antibodies (<b>B</b>; blue bars), should only block a SA independent pathway, there is still greater inhibition with Dd2 compared to 3D7. In the presence of both antibodies (<b>C</b>; anti-EBA-175_RII/rRH5 shown as red bars and anti-EBA-175_RII+anti-rRH5 shown as green bars, respectively), there is a significant difference in the ability of Dd2 to invade compared to 3D7, especially when individual antibodies are combined.</p

Invasion Inhibition of 3D7 with anti-EBA-175_RII and anti-rRH5 antibodies.

<p>Anti-EBA-175_RII (solid black line in panel <b>A</b>) and anti-rRH5 antibodies (solid blue line in panel <b>B</b>) inhibit invasion of 3D7 in a linear correlation to a similar extent. The two varying combinations used, anti-EBA-175_RII/anti-rRH5 and anti-EBA-175_RII+anti-rRH5 (solid red and solid green lines, respectively, in <b>C</b>), also showed the same positive correlation between increased antibody concentration and % inhibition (the color key for each antibody is conserved from <b>A</b>, <b>B</b>, and <b>C</b>). Percentage invasion inhibition from purified mouse IgG used as a control is shown as dashed black line (<b>A</b>, <b>B</b>, <b>C</b>).</p