A simple, ex vivo phagocytosis assay of Plasmodium vivax merozoites by flow cytometry

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.

鉴于吞噬作用（phagocytosis）是对抗疟疾的第一道防线，我们开发了一种针对间日疟原虫（Plasmodium vivax, P. vivax）裂殖子（merozoite）的吞噬检测方法，可用于候选疫苗的评估。具体流程简述如下：先通过注射器内装填的松散纤维素粉末去除白细胞，随后在E64蛋白酶抑制剂（E64 protease inhibitor）存在的条件下，使间日疟原虫滋养体（trophozoite）发育为富含裂殖子的裂殖体（schizont）阶段。经Percoll密度梯度离心（Percoll gradient）富集的裂殖体经化学裂解以释放裂殖子；随后将这些裂殖子置于两种经针对间日疟原虫裂殖子表面蛋白1（Merozoite Surface Protein-1, MSP1）N端与C端的人源及鼠源抗体（对应Nterm-PvMSP1与MSP119）处理的吞噬细胞系中，开展调理素依赖（opsonin-dependent）的裂殖子吞噬检测。该检测方法简便高效，可作为常规吞噬检测手段，用于评估裂殖子阶段的候选疫苗。