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

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