Abstract
BackgroundFunctional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos) was used for obtaining reliable live parasite counts through flow cytometry.MethodsBoth asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised parasites (induced by either medium starvation or by anti-malarial drug treatment) was carried out. Finally, parasite counts obtained by CMXRos staining through flow cytometry were used to determine specific growth inhibition index (SGI) in an antibody-dependent cellular inhibition (ADCI) assay.ResultsMitotracker Red CMXRos can reliably detect live intra-erythrocytic stages of P. falciparum. Comparison between staining of live with compromised parasites shows that CMXRos predominantly stains live parasites with functional mitochondria. Parasite counts obtained by CMXRos staining and flow cytometry were highly reproducible and can reliably determine the ability of IgG from hyper-immune individuals to inhibit parasite growth in presence of monocytes in ADCI assay. Further, a dose-dependent parasite growth inhibitory effect could be detected for both total IgG purified from hyper-immune sera and affinity purified IgGs against the N-terminal non-repeat region of GLURP in ADCI assays coupled with determination of parasite counts through CMXRos staining and flow cytometry.ConclusionsA flow cytometry method based on CMXRos staining for detection of live parasite populations has been optimized. This is a rapid and sensitive method with high inter-assay reproducibility which can reliably determine the anti-parasite effect mediated by antibodies in functional in vitro assays such as ADCI assay.
Highlights
Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates
Mitotracker Red CMXRos staining can reliably differentiate between uninfected and plasmodium falciparum parasitized erythrocytes In order to investigate the usefulness of Mitotracker Red CMXRos for differentiation between uninfected and parasitized erythrocytes, an asynchronous culture of P. falciparum NF54 was stained with CMXRos dye
Determination of parasitaemia by CMXRos-stained flow cytometry and Giemsa-stained microscopic counts for both early rings and mature trophozoites populations ranging between 1-8% parasitaemia were compared in Bland-Altman test to assess the agreement between the two methods (Figure 2A and B)
Summary
Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. Druilhe and co-workers have hypothesized that Plasmodium falciparum-specific antibodies can co-operate with human blood monocytes to control parasite multiplication in vivo and have developed the in vitro correlate of this immune effector mechanism – the antibody-dependent cellular inhibition (ADCI) assay [2]. When tested individually in Phase-1 clinical trials in malarianaïve volunteers, both antigens were found to elicit antibodies capable of mediating parasite killing in in vitro ADCI assays [10,11]. These findings have led to the production and clinical testing of a chimeric protein, GMZ2, containing both MSP3 and GLURP domains [12,13,14,15]. A robust and high throughput method for assessment of ADCI activity is highly desirable for evaluation of clinical trial samples from large Phase 2 efficacy trials
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