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

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 bloodstage 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 S = 1.07 95%CI : 1.03, 1.19. 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. Malaria remains a global health problem with over 200 million cases and more than 400,000 deaths annually 1. Most of these deaths are caused by the most virulent parasite Plasmodium falciparum. Ongoing interventions with insecticides, bed nets and artemisinin combination therapy had led to a decline of mortality and morbidity; however, the decline has stalled in recent years. It is still the hope that a malaria vaccine will facilitate the much-needed step towards eradication of the disease, especially in Sub-Saharan Africa where it is most relevant. There are many malaria vaccine candidates in development; these are targeted at the pre-erythrocytic-stage,

Reduced blood-stage malaria growth and immune correlates in humans following RH5 vaccination

“…A second strategy to improve RH5-based vaccines would be to incorporate the other, more recently described antigens CyRPA and RIPR, which associate with RH5 to form a heterotrimeric protein complex; like RH5, both of these antigens are highly conserved and essential and can induce high levels of growthinhibitory antibodies following vaccination of animals. 6,52 Finally, rational improvements of the design and delivery of the RH5 immunogen should also be explored. Strategies to achieve substantial improvements in the quantity and/or quality of vaccine-induced polyclonal anti-RH5 IgG would include analysis of vaccine-induced anti-RH5 human mAbs 53 to inform structure-based vaccine design 54 coupled with improved delivery of novel RH5 immunogen arrays on virus-like particles.…”

Strain-Dependent Inhibition of Erythrocyte Invasion by Monoclonal Antibodies Against Plasmodium falciparum CyRPA

“…This is due to PfCyRPA being a highly conserved target that participates in a non-redundant invasion pathway ( 9 , 14 ). Additionally, P. falciparum merozoite antigens, PfMSRP5, PfSERA9, PfRAMA, PfRipr and PfRh5 are able to induce growth inhibitory antibodies that in combination with anti-PfCyRPA antibodies have synergistic interactions in vitro ( 19 , 22 , 23 ). This suggests that combinations of merozoite antigens could be used in a rationally designed malaria vaccine to elicit synergistic polyclonal antibody responses.…”

Antibodies from malaria-exposed Malians generally interact additively or synergistically with human vaccine-induced RH5 antibodies

“…Bliss's and/or Loewe's definitions of additivity have been used in many studies, 13,15,34,38,40 whereas other studies used neither definition. 16,39 Bliss and Loewe additivity (and synergy) have different definitions, and both have advantages and disadvantages (a detailed mathematical discussion can be found elsewhere 41 ). Bliss additivity can be determined by testing a minimum of three experimental conditions (e.g., antibody A alone at one concentration, antibody B alone at one concentration, and a mixture of the two), but this definition carries a risk of self-synergy or self-antagonism (as described in detail previously 41 ).…”

“…However, to determine Loewe additivity/synergy, multiple combination GIAs are required for each antibody mixture, because each of the two antibodies must be tested at multiple concentrations. 41 Because limited amounts of human IgGs (especially RH5-specific IgGs) were available in this study, we designed our study to determine the interactions based on Bliss's definition.…”