Investigating Factors Influencing Variation in P. Falciparum Invasion Phenotyping Assays

Abstract

Malaria caused by P. falciparum, is the most life-threatening human parasitic disease, claiming over 400,000 deaths from about 200 million cases in 2017. Despite the yet unsuccessful efforts in the quest for an effective malaria vaccine, antigens expressed in the erythrocytic stage of P. falciparum represent highly promising vaccine candidates given their natural exposure to the immune system. Understanding the mechanism of erythrocyte invasion by P. falciparum mainly during active clinical infection is an important step towards developing an effective vaccine to counter the deleterious outcomes of this parasitic infection. However, comparison of pioneering studies assessing the invasion phenotypic diversity of P. falciparum is precluded by the lack of consensus in the protocols used in individual studies. As such, efforts towards the development of standard protocols necessitate the assessment of the impact of potential sources of variation in the measured outcome. Here, the effect of short-term culture adaptation on the invasion phenotypes of P. falciparum clinical isolates was assessed using freshly isolated parasites from children presenting an acute malaria illness. We also assessed the effect of short-term cryopreservation on the parasites’ invasion phenotype relative to the ex vivo invasion phenotype of the isogenic parasites. The effect of blood donor variability and some erythrocyte phenotypic features in the parasites’ invasion phenotype were also assessed here. Furthermore, we assessed the invasion phenotypic diversity of P. falciparum clinical isolates from four areas of varying transmission intensity in Ghana and compared the expression levels of the major parasites’ invasion-related ligands across isolates. Finally, we tested the invasion inhibitory activity of antibodies against two novel P. falciparum antigens and assessed the naturally-derived antibody responses against both antigens in children presenting at the hospital with symptomatic malaria. We showed that short-term cryopreservation has a minimal effect on the parasites’ invasion phenotype while short-term culture adaptation was found to affect the genotypic diversity and, to a lesser extent, the phenotypic diversity of P. falciparum clinical isolates. Blood donor variability was also found to affect P. falciparum invasion efficiency. However, this variation was associated with none of the erythrocyte phenotypic features tested here (e.g. blood group, hemoglobin genotype, receptor density). The erythrocyte invasion phenotyping experiments showed the predominance of sialic acid independent invasion pathway in Ghanaian clinical isolates and a high dependency on the CR1/PfRh4 mediated invasion pathway across all isolates. Moreover, invasion into trypsin-treated erythrocytes was found to be strongly correlated with the invasion into chymotrypsin-treated erythrocytes. The assessment of the transcript levels of invasion-related genes showed significantly higher expression levels of PfEBA family genes relative to the PfRh family genes, while the pattern of expression was similar across sites. Further analysis revealed a strong negative correlation between PfEBA175 and all other genes except for PfEBA140. Furthermore, the growth inhibition assays showed different ranges of dose-dependent inhibitory activities of individual antibodies against the target antigens used in his study. However, the combination of antibodies against different antigens showed no synergistic invasion inhibitory activities across all isolates. Finally, the level of antibody responses against the different antigens were strongly associated with transmission intensity. Taken together, our findings showed that differences in protocols may account for most of the variations in previously reported invasion phenotyping data across different countries. Therefore, this work has provided preliminary data upon which more rigorous experimental design should be based on for the optimization and establishment of standardized invasion phenotyping assays, which is critical for large-scale studies.