Abstract
As phagocytosis is the first line of defense against malaria, we developed a phagocytosis assay with Plasmodium vivax (P. vivax) merozoites that can be applied to evaluate vaccine candidates. Briefly, after leukocyte removal with loosely packed cellulose powder in a syringe, P. vivax trophozoites matured to the merozoite-rich schizont stages in the presence of the E64 protease inhibitor. The Percoll gradient-enriched schizonts were chemically disrupted to release merozoites that were submitted to merozoite opsonin-dependent phagocytosis in two phagocytic lines with human and mouse antibodies against the N- and C-terminus of P. vivax Merozoite Surface Protein-1 (Nterm-PvMSP1 and MSP119). The resulting assay is simple and efficient for use as a routine phagocytic assay for the evaluation of merozoite stage vaccine candidates.
Highlights
Based on Plasmodium falciparum studies, merozoite opsonisation appears to be correlated to immunity against malaria, and such merozoite phagocytosis assays could be useful to aid preclinical vaccine development and evaluate vaccine clinical trials.[1,2,3] Merozoite phagocytosis has never been evaluated in P. vivax, we adapted standardised protocols to develop a merozoite phagocytosis assay with saponin-treated P. vivax schizonts concentrated from clinical isolates, the flow cytometry was a useful tool for studying phagocytic uptake of blood stages.[4,5,6] The resulting assay is a simple to evaluate opsonising antibodies from malaria vaccine candidates
P. vivax trophozoites were matured in 20% hematocrit in 7.5% glucose McCoy medium supplemented with 10% AB+ serum at 5% O2, 5% CO2 and 90% N2 until the beginning of schizogony, according to previous study.[11]. After 24-30 h of culture, parasite-infected erythrocytes were treated with trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E64), a cysteine protease inhibitor, to ensure a maximum output of merozoite-rich schizonts, with some modifications.[12]. E64 ensured that the schizonts were fully mature after 46 h of culture and osmotically ruptured schizonts to release fully formed merozoites
Free merozoites and ruptured schizonts were incubated with mouse anti-N-term PvMSP1 and anti-MSP119 antibodies in bovine serum albumin (BSA)-phosphate buffer in 1.5 mL micro tubes for 30 min at room temperature and revealed with Alexa Flu
Summary
Based on Plasmodium falciparum studies, merozoite opsonisation appears to be correlated to immunity against malaria, and such merozoite phagocytosis assays could be useful to aid preclinical vaccine development and evaluate vaccine clinical trials.[1,2,3] Merozoite phagocytosis has never been evaluated in P. vivax, we adapted standardised protocols to develop a merozoite phagocytosis assay with saponin-treated P. vivax schizonts concentrated from clinical isolates, the flow cytometry was a useful tool for studying phagocytic uptake of blood stages.[4,5,6] The resulting assay is a simple to evaluate opsonising antibodies from malaria vaccine candidates. The reactivity of the eluted human antibodies and mouse immunised sera against anti-N-term PvMSP1 and anti-MSP119 were tested using enzymatic immune assays with the respective proteins. Free merozoites and ruptured schizonts were incubated with mouse anti-N-term PvMSP1 and anti-MSP119 antibodies in BSA-phosphate buffer in 1.5 mL micro tubes for 30 min at room temperature and revealed with Alexa Flu-
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