Merozoite surface proteins in red blood cell invasion, immunity and vaccines against malaria

Abstract

Malaria accounts for an enormous burden of disease globally, with Plasmodium falciparum accounting for the majority of malaria, and P. vivax being a second important cause, especially in Asia, the Americas and the Pacific. During infection with Plasmodium spp., the merozoite form of the parasite invades red blood cells and replicates inside them. It is during the blood-stage of infection that malaria disease occurs and, therefore, understanding merozoite invasion, host immune responses to merozoite surface antigens, and targeting merozoite surface proteins and invasion ligands by novel vaccines and therapeutics have been important areas of research. Merozoite invasion involves multiple interactions and events, and substantial processing of merozoite surface proteins occurs before, during and after invasion. The merozoite surface is highly complex, presenting a multitude of antigens to the immune system. This complexity has proved challenging to our efforts to understand merozoite invasion and malaria immunity, and to developing merozoite antigens as malaria vaccines. In recent years, there has been major progress in this field, and several merozoite surface proteins show strong potential as malaria vaccines. Our current knowledge on this topic is reviewed, highlighting recent advances and research priorities.

Keywords: Plasmodium falciparum; Plasmodium vivax; antibodies; immunity; invasion; merozoites; vaccines.

Figure 1.

‘Invasion of RBC by P. falciparum merozoites.’ (A) After release from schizonts, most merozoites are thought to invade RBCs within several minutes, although some may take substantially longer. Invasion commences with initial, or primary, reversible attachment of the merozoite to the RBC surface. The merozoite reorientates, if needed, so that it's apical end makes contact with the RBC surface. Secondary interactions then occur, mediating strong and irreversible attachment to the RBC, leading to the release of contents from the rhoptries and the formation of the tight junction. Merozoite invasion then proceeds via an actin-myosin motor, and processing of many merozoite surface proteins occurs. Invasion is completed by resealing of the RBC membrane, and completion of the processing and shedding of merozoite surface proteins (image modified from Richards and Beeson 2009). (B) Electron micrographs showing different stages in RBC invasion by a merozoite (from Boyle et al.2010b).

Figure 2.

‘Processing of merozoite proteins before, during, and after invasion.’ (A) Prior to invasion, PfSUB1 is released from the merozoite into the parasitophorous vacuole lumen where it processes SERA proteins and a number of merozoite surface proteins. This activates SERA proteins so they can mediate rupture, and matures some surface proteins to functional conformations (e.g. MSP1) (B) Around the time of rupture, PfSUB2 is released and translocates to the apex of the merozoite. PfSUB2 cleaves MSP1-42, AMA1 and PTRAMP. During invasion, cleaved and peripherally-associated surface proteins are shed at the point of tight junction, while other proteins such as MSP2 and MSP4 are internalized during invasion. PfROM1 and PfROM4 are also involved and cleave EBA and PfRH proteins, as well as AMA1. (C) Following invasion, MSP2 and possibly other proteins are rapidly degraded, whereas other proteins, including MSP1-19 and MSP4, are maintained post-invasion and may have roles in intraerythrocytic parasite development.

Figure 3.

‘Association between antibodies to P. falciparum merozoite antigens and protection from malaria.’ Antibodies to a range of different merozoite proteins were evaluated in a longitudinal cohort of children living in a malaria-endemic region of Papua New Guinea. Antibody responses were prospectively related to the risk of malaria over a 6-month period of follow-up; malaria was defined as parasitemia of greater than 5000 parasites/ul of blood and fever. In the figure, antigen-specific antibodies are ranked by the strength of their association with protection (determined from hazard ratios calculated using the Cox proportional hazards model). The red line indicates no protective association. Error bars represent the 95% confidence interval. Figure was adapted from Richards et al. (2013).

Figure 4.

‘Antibody-mediated mechanisms of immunity to merozoites.’ (A) Antibodies to merozoites may mediate immunity through a number of mechanisms. This includes the ability of antibodies to directly inhibit invasion of merozoites, interact with complement (red stars) to inhibit invasion or lyse merozoites, and agglutinate merozoites to inhibit their dispersal after egress from schizonts. Opsonization of merozoites by antibodies promotes their phagocytosis by monocytes and macrophages, and killing by neutrophils. Phagocytosis of opsonized merozoites by monocytes results in activation and the production of TNF-alpha and other cytokines, and secretion of soluble factors (represented by triangles) that inhibit parasite growth (referred to as ADCI). Different antibody types (such as different IgG subclasses) may have different functional activities, particularly for complement fixation and Fc–receptor interactions; however, these differences are not currently clearly defined. To reflect these potentially important differences, we have shown antibodies as yellow or green to represent the presence of different antibody types that can mediate functional activities. (B) Early stages of phagocytosis of antibody-opsonized merozoites by a THP1 monocyte imaged using scanning electron microscopy (adapted from (Osier et al.2014a)). (C) Antibodies to merozoites promote the activation of complement on the merozoite surface by fixing complement component C1q leading to the activation of complement through the classical cascade. A key step in the cascade is the formation of C3b, which is labelled in this figure with gold particles on the surface of merozoites by transmission electron microscopy.

Figure 5.

‘Use of alternate invasion pathways and immune evasion.’ (A) In this example of alternate invasion pathways, the clonal parasite isolate gives rise to merozoites that largely use a sialic acid-dependent invasion pathway (blue merozoites); a small subpopulation of merozoites use an alternate sialic acid independent pathway (yellow merozoites). In the absence of any selective pressure, parasite replication gives rise to a population of merozoites that predominantly invade via a sialic acid dependent invasion. Selective pressure on invasion, either through immune selection (e.g. antibodies that block ligands of sialic acid dependent invasion) or phenotypic selection (invasion into RBCs with deficiency in sialic acid receptors) leads to the emergence of parasites using an alternate sialic acid independent invasion pathway. The use of an alternate pathway can also be generated artificially by genetic disruption of EBA175. The sialic acid dependent invasion pathway uses the EBA ligands and PfRH1, whereas the sialic acid independent invasion pathway has a greater reliance on PfRH4 and PfRH2. (B) Differential inhibition by antibodies from malaria-exposed individuals of P. falciparum lines using different invasion pathways. In these examples, changes in invasion pathways were generated by deletion of EBA175, EBA140 or EBA181. Results show the proportion of samples (n = 130) that differentially inhibited the invasion of the parental parasites compared to parasites with disruption of specific EBA genes. No difference in inhibition between the isolates was regarded as <25% difference in the level of inhibition (indicated in blue). The proportion of samples that inhibited the knockout line more than the 3D7wt line (by >25%) is shown in green; the proportion of samples that inhibited 3D7wt more than the knockout line (by >25%) is shown in red. Data reproduced from (Persson et al.2013).