Development of vaccines for malaria remains a high priority in the effort to control malaria worldwide. Blood-stage vaccines are important components of these efforts and functional
in vitro assays are particularly needed to facilitate the clinical evaluation of candidate vaccines and possibly for future down-selection of vaccine candidates. The antibody-dependent cellular inhibition (ADCI) assay may provide one such tool [
1]. 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 accordingly developed the
in vitro correlate of this immune effector mechanism – the antibody-dependent cellular inhibition (ADCI) assay [
2]. Immune-epidemiological studies support the
in vivo relevance of a monocyte-dependent, antibody-mediated mechanism by showing a correlation between the acquisition of clinical immunity and levels of cytophilic IgG subclasses (IgG1 and IgG3) [
3,
4]. Cytophilic antibodies opsonize merozoites and activate the monocyte by binding FcγIIa/FcγIIIa present on its cell surface [
5,
6]. Upon activation, monocytes are thought to release TNF, and other as yet uncharacterized factor(s), that inhibit intra-erythrocytic parasite growth [
5,
7]. Among the various targets of ADCI-effective antibodies, merozoite surface protein 3 (MSP3) and the glutamate-rich protein (GLURP) have been extensively studied. Affinity-purified IgG to MSP3 and GLURP obtained from endemic sera, have significantly reduced parasite growth in
in vitro ADCI assay [
8,
9]. When tested individually in Phase-1 clinical trials in malaria-naïve 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‐
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.
Historically,
P. falciparum growth
in vitro has been monitored by microscopy, radioactive hypoxanthine uptake [
16], and by an enzyme-based method [
17]. Radioactive labelling and enzyme-based methods can be problematic in ADCI assays, which include monocytes together with parasitized erythrocytes, as monocytes could contribute to the readout. Microscopic examination of Giemsa-stained slides, therefore, remains the ‘gold standard’ for the quantification of blood-stage malaria parasites in ADCI assays. However, microscopic evaluation is time-consuming and relies on the skills of the microscopists trained perfectly to identify the different parasite stages and to distinguish between live and compromised parasites [
18]. These shortcomings are of particular concern, since inter-reader variability gives rise to the common criticism that microscopy is relatively subjective [
19,
20]. Thus, there is a need for an improved readout for parasite counts in the ADCI assay. Parasite quantification based on flow cytometry has been proposed with the goal of increasing accuracy and reducing subjectivity. Different permeable nucleic acid binding dyes such as Hoechst 33258, 33342 [
21], SYBR Green I [
22‐
24], thiazole orange [
25], acridine orange [
26], ethidium bromide [
27], hydroethidine [
28,
29], SYTO-16 [
30], or propidium iodide [
31] have been used for enumeration of infected erythrocytes. However, these dyes except possibly hydroethidine, are unable to distinguish between live and compromised parasites as they could stain remnant DNA and/or RNA in compromised parasites. Mitochondrial membrane potential is a key indicator of cellular viability since it reflects metabolic activity and integrity [
32]. Dyes which only bind the polarized mitochondrial membrane have been developed to differentiate between live and compromised cells [
33]. However, these dyes have inherent problems as rhodamine 123 is highly susceptible to photo-bleaching and exhibits strong photo-induced toxicity, and JC-1 is not only specific for mitochondria [
34]. Moreover, both dyes display reversible binding to the polarized membrane leading to potential losses during sample preparation for flow cytometry. Further, rhodamine dyes have adverse effects on mitochondrial respiration [
35,
36] and do not appear to be strictly dependent on mitochondrial membrane potential for intramitochondrial accumulation. Their use in careful studies on mitochondrial physiology is therefore problematic. Mitotracker Red CMXRos dye has the following advantages: it is intrinsically fluorescent, binds irreversibly to the polarized mitochondrial membrane, and does not require reduction or oxidation for emission of fluorescence [
37]. CMXRos dye has an alkylating chloromethyl group, which can react with accessible nucleophiles, including thiol groups of peptides and proteins, to form aldehyde-fixable conjugates. The covalent binding to the polarized inner mitochondrial membrane enhances dye retention during washing and fixation of cells, thereby making this dye particularly suited for flow cytometry and fluorescence microscopy [
38]. Of the above-mentioned dyes, hydroethidine [
29,
39] and rhodamine 123 [
40] have previously been used in flow cytometry-based readouts for parasite counts in ADCI assays. In this study, a protocol for flow cytometric evaluation of CMXRos stained parasitized erythrocytes was developed and compared with microscopy. It was concluded that the CMXRos staining-based flow cytometric protocol is a rapid and accurate tool for assessing parasitaemia in the ADCI assay.