Malaria Vaccine Development: Focusing Field Erythrocyte Invasion Studies on Phenotypic Diversity: The West African Merozoite Invasion Network (WAMIN)

Abstract

Erythrocyte invasion by Plasmodium falciparum merozoites is an essential step for parasite survival and proliferation. Invasion is mediated by multiple ligands, which could be promising vaccine targets. The usage and sequence of these ligands differs between parasites, yet most studies of them have been carried out in only a few laboratory-adapted lines. To understand the true extent of natural variation in invasion phenotypes and prioritize vaccine candidates on a relevant evidence base, we need to develop and apply standardized assays to large numbers of field isolates. The West African Merozoite Invasion Network (WAMIN) has been formed to meet these goals, expand training in Plasmodium phenotyping, and perform large-scale field phenotyping studies in order to prioritize blood stage vaccine candidates.

Keywords: alternative receptors; antibody inhibition assays; invasion; malaria; merozoite; vaccine.

Figure 1.. Erythrocyte Invasion is a Complex Process Involving Multiple Steps.

(A) Mature merozoites are released from the bursting schizont, and disperse among surrounding red blood cells. Merozoites initially contact, probably at random, uninfected erythrocytes and loosely adhere on their cell surface. (B) Primary merozoite contact is associated with an obvious warping of erythrocyte cell membrane during which the merozoite reorients itself to place its apical end in direct apposition with the erythrocyte cell membrane [49]. Merozoite reorientation is followed by the establishment of additional protein–protein contacts between the two juxtaposed cell membranes, probably committing the merozoite to invasion [–52]. (C) These contacts are followed by the formation of a so-called tight or moving junction, which is transformed into a migrating ring (the moving junction) that moves towards the basal end of merozoite, moving the merozoite into the erythrocyte [14,53]. The function of ligands at each step is not completely well established, although the erythrocyte binding-like (EBL) and reticulocyte binding protein homolog (RH) ligands are believed to bind to specific erythrocyte receptors at some point between the initial merozoite attachment and the formation of the tight junction [14,52]. Merozoite thrombospondin-related anonymous protein (MTRAP) is thought to bridge the gap between parasite actin–myosin motor and host erythrocyte plasma membrane during the moving junction step [54,55], which is mediated by the apical membrane antigen (AMA1)–rhoptry neck protein (RON2/4/5) complex.

Figure 2.. Previous Studies Using Enzymatic Treatment of Erythrocytes to Examine Invasion Phenotypes of Plasmodium falciparum Clinical Isolates.

(A) Global distribution of the ten different endemic countries where erythrocyte invasion phenotypes have been determined. Phenotypes of 536 clinical P. falciparum isoaltes have been reported in 13 different research papers; number of isolates studied from each country are shown in parentheses [,,,–,,–40]. A large majority of the isolates (437, 81.5% of the total) were from patients in West Africa. (B) Summary of the seven studies [17,23,32,34,37,38,40] that have investigated erythrocyte invasion phenotypes of West African clinical P. falciparum isolates. The data highlight the differences in numbers of isolates tested, and concentrations (per ml) of neuraminidase (Neu), chymotrypsin (Chy), and trypsin (Try) used for erythrocyte treatment. Senegal 1 [37]; Senegal 2 [32]; Senegal 3 [23]; Gambia 1 [34]; Gambia 2 [38]; Ghana 1 [17]; Guinea, Senegal 4, Ghana 2 [40].

Figure 3.. Example of a Merozoite Invasion Assay Protocol.

Erythrocytes to be invaded (acceptor cells, blue) are fluorescently labeled with DDAO-SE and treated with combinations of the standard enzymes used for merozoite invasion phenotyping – trypsin (Try), chymotrypsin (Chy), and neuraminidase (Neu) – or left untreated [56]. Acceptor cells are then incubated with parasitized erythrocytes (donor cells, red) that have been rendered nonpermissive for invasion by enzymatic treatment. If desired, antibodies against specific receptors or ligands may be included to determine their inhibitory activities. Parasites are allowed to invade over 48 h in culture, then erythrocytes are stained with SYBR Green I to label parasite DNA and analyzed by flow cytometry. Acceptor erythrocytes that have been invaded are identified as DDAO-SE-positive and SYBR Green I-positive cells. Even with enzyme treatment, there can be low levels of background invasion into donor cells or, alternatively, nonviable schizonts or gametocytes can be present; thus, distinguishing between parasites in donor and acceptor cell populations is crucial to measuring true reinvasion efficiency. Comparing invasion in untreated and enzyme-treated cells reveals the invasion phenotype of the parasites in the acceptor cells.