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. 2020 Jul 16;10(1):11802.
doi: 10.1038/s41598-020-67877-8.

Bliss' and Loewe's additive and synergistic effects in Plasmodium falciparum growth inhibition by AMA1-RON2L, RH5, RIPR and CyRPA antibody combinations

Yvonne Azasi #  1 , Shannon K Gallagher #  2 , Ababacar Diouf  1 , Rebecca A Dabbs  3 , Jing Jin  3 , Syed Yusuf Mian  4 , David L Narum  5 , Carole A Long  1 , Deepak Gaur  4 , Simon J Draper  3 , Michael P Fay  6 , Louis H Miller  7 , Kazutoyo Miura  8
Affiliations

Bliss' and Loewe's additive and synergistic effects in Plasmodium falciparum growth inhibition by AMA1-RON2L, RH5, RIPR and CyRPA antibody combinations

Yvonne Azasi et al. Sci Rep. .

Abstract

Plasmodium invasion of red blood cells involves malaria proteins, such as reticulocyte-binding protein homolog 5 (RH5), RH5 interacting protein (RIPR), cysteine-rich protective antigen (CyRPA), apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2), all of which are blood-stage malaria vaccine candidates. So far, vaccines containing AMA1 alone have been unsuccessful in clinical trials. However, immunization with AMA1 bound with RON2L (AMA1-RON2L) induces better protection against P. falciparum malaria in Aotus monkeys. We therefore sought to determine whether combinations of RH5, RIPR, CyRPA and AMA1-RON2L antibodies improve their biological activities and sought to develop a robust method for determination of synergy or additivity in antibody combinations. Rabbit antibodies against AMA1-RON2L, RH5, RIPR or CyRPA were tested either alone or in combinations in P. falciparum growth inhibition assay to determine Bliss' and Loewe's additivities. The AMA1-RON2L/RH5 combination consistently demonstrated an additive effect while the CyRPA/RIPR combination showed a modest synergistic effect with Hewlett's [Formula: see text] Additionally, we provide a publicly-available, online tool to aid researchers in analyzing and planning their own synergy experiments. This study supports future blood-stage vaccine development by providing a solid methodology to evaluate additive and/or synergistic (or antagonistic) effect of vaccine-induced antibodies.

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Conflict of interest statement

SJD is a named inventor on patent applications relating to RH5 and/or other malaria vaccines and immunization regimens. The other authors do not have conflict of interest.

Figures

Figure 1
Figure 1
Total IgGs from rabbits immunized with AMA1-RON2L, RIPR or CyRPA binds to their specific antigens. Antibody reactivity of 1 μg/mL of AMA1-RON2L, RIPR, CyRPA or control total IgG (two rabbit IgGs per antigen) to 1 μg/mL of recombinant AMA1, RON2 peptide, RIPR or CyRPA protein was tested in ELISA. Each dot represents the average OD value of each rabbit IgGs from two independent assays. RH5 antibody was not tested by ELISA as the IgG came from a previously published study.
Figure 2
Figure 2
Growth inhibition activity of AMA1-RON2L, RIPR, CyRPA and RH5 antibodies. Increasing concentrations of purified total IgG from rabbits immunized with RH5 (a), AMA1-RON2L (b), RIPR (c), CyRPA (d) or Control IgG (e) were tested for growth inhibition of 3D7 clone parasites. Data shown are the mean and SEM of at least two independent experiments each with three replicate wells. Dashed line shows 50% inhibition. For RH5 (a), GIA was done with a pool of total IgG from rabbits immunized with RH5. For the other antigens or negative control (b–e), two total IgG samples from two rabbits were tested individually (two lines per antigen).
Figure 3
Figure 3
Growth inhibition activity of RH5/AMA1-RON2L, RIPR/AMA1-RON2L, CyRPA/AMA1-RON2L or CyRPA/RIPR total IgGs combinations. A fixed concentration (0.078 mg/mL) of AMA1-RON2L antibody was mixed with various concentrations of RH5 (a), RIPR (b) or CyRPA (c) antibodies. For the CyRPA/RIPR combination (d), a fixed concentration of RIPR antibody was tested with various concentrations of CyRPA antibody. Data shown are from n ≥ 2 independent experiments, each with 2 or 3 replicate wells. % GIA (y-axis) means % inhibition determined by GIA with 3D7 clone. Shaded regions are 95% confidence intervals (95%CI) by the parametric bootstrap percentile method. The green-yellow band shows % GIA of the titrated IgG: RH5 (a), RIPR (b) or CyRPA (c and d), which was tested alone at the indicated concentrations; blue shows % GIA from the Bliss model assuming Bliss’ additivity (no interaction effect); and purple shows experimentally observed (plus 95%CI) % GIA from the mixture of the two IgGs. The grey band shows % GIA from the fixed concentration of the fixed IgG when tested alone; AMA1-RON2L (a–c) or RIPR (d).
Figure 4
Figure 4
Model fits. The median estimate % GIA (color lines) and pointwise 95% central quantiles (color ribbons) are shown with actual observed % GIA (black dots). The top line in each row refers to the titrated antibody (the test concentration is shown in x-axis) and the second line in each row is the antibody that is being fixed to a certain quantity (the concentration in mg/mL is shown next to the antibody name). The top two rows show the data of RH5/AMA1-RON2L combination, and the bottom rows for the CyRPA/RIPR combination.
Figure 5
Figure 5
Isobolograms for model fits. The ED50 line for observed dose-combinations (black) and the 95% confidence interval of the inverse Hewlett S statistic (blue line), which corresponds to equal doses (after scaling by the ED50) of the two concentrations are shown; (a) RH5/AMA1-RON2L combination, and (b) CyRPA/RIPR combination. Dashed red line shows null synergy/antagonism interaction, and the synergy/antagonism hypothesis is rejected when an inverse Hewlett S line intersects with the null line.
Figure 6
Figure 6
Power simulation in three scenarios. The power (%) of our model to detect significant interactions depending on the initial parameters are shown. Three sets of estimated initial parameters corresponding to the model estimates from the pairs of RH5/AMA1-RON2L (a), CyRPA/RIPR (b), and RH5/RH4 (c, *data from Williams et al.). (a) has an interaction effect of close to zero, (τ1= − 0.06), (b) has a modest synergy effect (τ1= 0.25), and (c) has a large synergy effect (τ1= 36). The x-axis is the number of concentrations for one of the antibodies tested in an assay. For example, grid of 3 means each antibody is tested at 3 different concentrations, totaling 3 × 3 = 9 conditions per assay. The y-axis is the number of times to repeat the assay.
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