Treatment Of Plasmodium Falciparum Merozoites With The Protease Inhibitor E64 And Mechanical Filtration Increases Their Susceptibility To Complement Activation

Plasmodium falciparummalaria killed 451,000 people in 2017. Merozoites, the stage of the parasite that invades RBCs, are a logical target for vaccine development. Treatment with the protease inhibitor E64 followed by filtration through a 1.2 mu m filter is being used to purify merozoites for immunologic assays. However, there have been no studies to determine the effect of these treatments on the susceptibility of merozoites to complement or antibodies. To address this gap, we purified merozoites with or without E64 followed by filtration through either a 1.2 or 2.7 mu m filter, or no filtration. Merozoites were then incubated in either 10% fresh or heat-inactivated serum followed by surface staining and flow cytometry with monoclonal antibodies against the complement effector molecules C3b or C5b9. To determine the effect of anti-merozoite antibodies, we incubated merozoites with MAb5.2, a mouse monoclonal antibody that targets the merozoite surface protein 1. We used an amine-reactive fluorescent dye to measure membrane integrity. Treatment with E64 resulted in an insignificant increase in the proportion of merozoites that were C3b positive but in a significant increase in the proportion that were C5b9 positive. Filtration increased the proportion of merozoites that were either C3b or C5b9-positive. The combination of filtration and E64 treatment resulted in marked deposition of C3b and C5b9. MAb5.2 induced greater complement deposition than serum alone or an IgG2b isotype control. The combination of E64 treatment, filtration, and MAb5.2 resulted in very rapid and significant deposition of C5b9. Filtration through the 1.2 mu m filter selected a population of merozoites with greater membrane integrity, but their integrity deteriorated rapidly upon exposure to serum. We conclude that E64 treatment and filtration increase the susceptibility of merozoites to complement and antibody. Filtered or E64-treated merozoites are not suitable for immunologic studies that address the efficacy of antibodiesin vitro.