The role of antibodies to Plasmodium falciparum-infected-erythrocyte surface antigens in naturally acquired immunity to malaria

Plasmodium falciparum, the most virulent species of human malaria parasite, causes 1-3 million deaths per year. Because this parasite is susceptible to naturally acquired host immunity the main burden of diseases falls on young children. The mechanism of this immunity is still unclear. However, the parasite makes a considerable investment in the insertion of highly polymorphic antigens (parasite-infected-erythrocyte surface antigens, PIESA) on the infected erythrocyte surface, and these antigens are potentially important immune targets.     

Trafficking of malarial proteins to the host cell cytoplasm and erythrocyte surface membrane involves multiple pathways

During the asexual stage of malaria infection, the intracellular parasite exports membranes into the erythrocyte cytoplasm and lipids and proteins to the host cell membrane, essentially "transforming" the erythrocyte. To investigate lipid and protein trafficking pathways within Plasmodium falciparum-infected erythrocytes, synchronous cultures are temporally analyzed by confocal fluorescence imaging microscopy for the production, location and morphology of exported membranes (vesicles) and parasite proteins. Highly mobile vesicles are observed as early as 4 h postinvasion in the erythrocyte cytoplasm of infected erythrocytes incubated in vitro with C6-NBD-labeled phospholipids. These vesicles are most prevalent in the trophozoite stage. An immunofluorescence technique is developed to simultaneously determine the morphology and distribution of the fluorescent membranes and a number of parasite proteins within a single parasitized erythrocyte. Parasite proteins are visualized with FITC- or Texas red-labeled monoclonal antibodies. Double-label immunofluorescence reveals that of the five parasite antigens examined, only one was predominantly associated with membranes in the erythrocyte cytoplasm. Two other parasite antigens localized only in part to these vesicles, with the majority of the exported antigens present in lipid-free aggregates in the host cell cytoplasm. Another parasite antigen transported into the erythrocyte cytoplasm is localized exclusively in lipid-free aggregates. A parasite plasma membrane (PPM) and/or parasitophorous vacuolar membrane (PVM) antigen which is not exported always colocalizes with fluorescent lipids in the PPM/PVM. Visualization of two parasite proteins simultaneously using FITC- and Texas red-labeled 2 degrees antibodies reveals that some parasite proteins are constitutively transported in the same vesicles, whereas other are segregated before export. Of the four exported antigens, only one appears to cross the barriers of the PPM and PVM through membrane-mediated events, whereas the others are exported across the PPM/PVM to the host cell cytoplasm and surface membrane through lipid (vesicle)-independent pathways.     

Lipid compartmentalization in erythrocytes parasitized by Plasmodium spp

Although reasonably well protected from the host immune system by the erythrocyte membrane, the intraerythrocytic malaria parasite has to make that membrane compatible with its own requirements for development and multiplication. The development of Plasmodium spp brings about major changes in the lipid composition of the host cell membrane, as well as in its physical properties. The parasite itself has a lipid composition that differs from that of the host cell and an intense lipid trafficking seems to occur between intracellular parasite and host cell membrane. Here, Ana Paula Sim?es, Ben Roelofsen and Jos Op den Kamp discuss how, despite serious methodological limitations and the existence of some conflicting results, an overall picture of lipid compartmentalization within the parasitized erythrocyte is perceived.     

Plasmodium fragile: detection of a ring-infected erythrocyte surface antigen (RESA)

A ring-infected erythrocyte surface antigen (RESA) has been detected by modified immunofluorescence assay in erythrocytes infected with the simian malaria parasite, Plasmodium fragile. This RESA, of Mr 95,000, shares many characteristics with the RESA initially found in the human malaria parasite P. falciparum. Both antigens are found in the membrane of erythrocytes infected with young asexual parasite stages, in merozoite-enriched preparations, and in parasite culture supernatant. Since the RESA of P. falciparum has been shown to confer protective immunity and since P. fragile infection of rhesus monkeys mimics P. falciparum infection in humans, the finding of a RESA in P. fragile underlines the importance of this species as an animal model for antimalarial vaccines.     

Malaria impairs T cell clustering and immune priming despite normal signal 1 from dendritic cells

Interactions between antigen-presenting dendritic cells (DCs) and T cells are essential for the induction of an immune response. However, during malaria infection, DC function is compromised and immune responses against parasite and heterologous antigens are reduced. Here, we demonstrate that malaria infection or the parasite pigment hemozoin inhibits T cell and DC interactions both in vitro and in vivo, while signal 1 intensity remains unaltered. This altered cellular behaviour is associated with the suppression of DC costimulatory activity and functional T cell responses, potentially explaining why immunity is reduced during malaria infection.     

Plasmodium lipid rafts contain proteins implicated in vesicular trafficking and signalling as well as members of the PIR superfamily, potentially implicated in host immune system interactions

Plasmodium parasites, the causal agents of malaria, dramatically modify the infected erythrocyte by exporting parasite proteins into one or multiple erythrocyte compartments, the cytoplasm and the plasma membrane or beyond. Despite advances in defining signals and specific cellular compartments implicated in protein trafficking in Plasmodium-infected erythrocytes, the contribution of lipid-mediated sorting to this cellular process has been poorly investigated. In this study, we examined the proteome of cholesterol-rich membrane microdomains or lipid rafts, purified from erythrocytes infected by the rodent parasite Plasmodium berghei. Besides structural proteins associated with invasive forms, we detected chaperones, proteins implicated in vesicular trafficking, membrane fusion events and signalling. Interestingly, the raft proteome of mixed P. berghei blood stages included proteins encoded by members of a large family (bir) of putative variant antigens potentially implicated in host immune system interactions and targeted to the surface of the host erythrocytes. The generation of transgenic parasites expressing BIR/GFP fusions confirmed the dynamic association of members of this protein family with membrane microdomains. Our results indicated that lipid rafts in Plasmodium-infected erythrocytes might constitute a route to sort and fold parasite proteins directed to various host cell compartments including the cell surface.     

Morphological changes in erythrocytes induced by malarial parasites

Host cell alterations induced by Plasmodium falciparum, P. brasilianum, P. vivax and P. malariae were described by electron microscopy and post-embedding immunoelectron microscopy. P. falciparum infection induces knobs, electron-dense material and clefts in the erythrocyte. Clefts are involved in exporting P. falciparum antigen from the parasite to the erythrocyte membrane. P. falciparum antigen is present in knobs which adhere to endothelial cells causing the blockage of cerebral capillaries and ensuing pathological changes in cerebral tissues. P. brasilianum infection induces knobs, short and long clefts and electron-dense material. These structures appear to contain different P. brasilianum antigens. This indicates that each structure functions independently in trafficking P. brasilianum protein to the erythrocyte surface. P. vivax infection induces caveola-vesicle complexes and clefts in the erythrocyte. These structures are also involved in trafficking P. vivax protein from the parasite to the erythrocyte membrane. P. malariae induces caveolae, electron-dense material, vesicles, clefts and knobs in the erythrocyte. Although vesicles and caveolae are seen in the erythrocyte cytoplasm, they do not form caveola-vesicle complexes as seen in P. vivax-infected erythrocytes. They also appear to be involved in trafficking of malaria antigens. These studies, therefore, indicate that host cell changes occur in order to facilitate the transport of malarial antigens to the host cell membrane. The significance of these phenomena is still not clear.     

Natural Regulatory T Cells in Malaria: Host or Parasite Allies?

Plasmodium falciparum malaria causes 500 million clinical cases with approximately one million deaths each year. After many years of exposure, individuals living in endemic areas develop a form of clinical immunity to disease known as premunition, which is characterised by low parasite burdens rather than sterilising immunity. The reason why malaria parasites persist under a state of premunition is unknown but it has been suggested that suppression of protective immunity might be a mechanism leading to parasite persistence. Although acquired immunity limits the clinical impact of infection and provides protection against parasite replication, experimental evidence indicates that cell-mediated immune responses also result in detrimental inflammation and contribute to the aetiology of severe disease. Thus, an appropriate regulatory balance between protective immune responses and immune-mediated pathology is required for a favourable outcome of infection. As natural regulatory T (T_reg) cells are identified as an immunosuppressive lineage able to modulate the magnitude of effector responses, several studies have investigated whether this cell population plays a role in balancing protective immunity and pathogenesis during malaria. The main findings to date are summarised in this review and the implication for the induction of pathogenesis and immunity to malaria is discussed.     

Vesicle-mediated trafficking of parasite proteins to the host cell cytosol and erythrocyte surface membrane in Plasmodium falciparum infected erythrocytes

During the development of the asexual stage of the malaria parasite, Plasmodium falciparum, the composition, structure and function of the host cell membrane is dramatically altered, including the ability to adhere to vascular endothelium. Crucial to these changes is the transport of parasite proteins, which become associated with or inserted into the erythrocyte membrane. Protein and membrane targeting beyond the parasite plasma membrane must require unique pathways, given the parasites intracellular location within a parasitophorous vacuolar membrane and the lack of organelles and biosynthetic machinery in the host cell necessary to support a secretory system. It is not clear how these proteins cross the parasitophorous vacuolar membrane or how they traverse the erythrocyte cytosol to reach their final destinations. The identification of: (1) a P. falciparum homologue of the protein Sar1p, which is an essential component of the COPII-based secretory system in mammalian cells and yeast and (2) electron-dense, possibly coated, secretory vesicles bearing P. falciparum erythrocyte membrane protein 1 and P. falciparum erythrocyte membrane protein 3 in the host cell cytosol of P. falciparum infected erythrocytes recently provided the first direct evidence of a vesicle-mediated pathway for the trafficking of some parasite proteins to the erythrocyte membrane. The major advance in uncovering the parasite-induced secretory pathway was made by incubating infected erythrocytes with aluminium tetrafluoride, an activator of guanidine triphosphate-binding proteins, which resulted in the accumulation of the vesicles into multiple vesicle strings. These vesicle complexes were often associated with and closely abutted the erythrocyte membrane, but were apparently prevented from fusing by the aluminium fluoride treatment, making their capture by electron microscopy possible. It appears that malaria parasites export proteins into the host cell cytosol to support a vesicle-mediated protein trafficking pathway.     

Immune response to pre-erythrocytic stages of malaria parasites

Immunization with radiation-attenuated Plasmodium spp. sporozoites induces sterile protective immunity against parasite challenge. This immunity is targeted primarily against the intrahepatic parasite and appears to be sustained long term even in the absence of sporozoite exposure. It is mediated by multifactorial mechanisms, including T cells directed against parasite antigens expressed in the liver stage of the parasite life cycle and antibodies directed against sporozoite surface proteins. In rodent models, CD8+ T cells have been implicated as the principal effector cells, and IFN-gamma as a critical effector molecule. IL-4 secreting CD4+ T cells are required for induction of the CD8+ T cell responses, and Th1 CD4+ T cells provide help for optimal CD8+ T cell effector activity. Components of the innate immune system, including gamma-delta T cells, natural killer cells and natural killer T cells, also play a role. The precise nature of pre-erythrocytic stage immunity in humans, including the contribution of these immune responses to the age-dependent immunity naturally acquired by residents of malaria endemic areas, is still poorly defined. The importance of immune effector targets at the pre-erythrocytic stage of the parasite life cycle is highlighted by the fact that infection-blocking immunity in humans rarely, if ever, occurs under natural conditions. Herein, we review our current understanding of the molecular and cellular aspects of pre-erythrocytic stage immunity.     

Human immune responses against sexual stages of malaria parasites: considerations for malaria vaccines

Studies on the natural immune responses to the sexual stages of malaria parasites have been reviewed in the context of human malaria transmission-blocking vaccines. Antibodies against the sexual stages of the malaria parasite, gametocytes and gametes, are readily evoked by natural malaria infections. These antibodies that suppress infectivity at high concentrations can, at low concentrations, enhance the development of the parasite in the mosquito; however, because enhancing antibodies are prevalent during natural malaria infections, it is likely that a vaccine would rapidly boost these antibodies to blocking levels. The immunogenicity of sexual stage antigens appears to be constrained in the human host, probably due to T epitope polymorphism and MHC restriction in humans. These constraints apply mainly to those antigens that are sensitive targets of host immunity such as the gamete surface antigens and not to internal gamete antigens, indicating that antigenic polymorphism may have evolved in response to immune selection pressure. Evidence for immunosuppression of the host by exposure to endemic malaria is presented and its consequences on vaccine development are discussed.     

Whole-genome analysis of Malawian Plasmodium falciparum isolates identifies possible targets of allele-specific immunity to clinical malaria

Individuals acquire immunity to clinical malaria after repeated Plasmodium falciparum infections. Immunity to disease is thought to reflect the acquisition of a repertoire of responses to multiple alleles in diverse parasite antigens. In previous studies, we identified polymorphic sites within individual antigens that are associated with parasite immune evasion by examining antigen allele dynamics in individuals followed longitudinally. Here we expand this approach by analyzing genome-wide polymorphisms using whole genome sequence data from 140 parasite isolates representing malaria cases from a longitudinal study in Malawi and identify 25 genes that encode possible targets of naturally acquired immunity that should be validated immunologically and further characterized for their potential as vaccine candidates.     

Working without a blindfold: the critical role of diagnostics in malaria control

Background

The insertion of parasite antigens into the host erythrocyte membrane and the structure and distribution of Plasmodium falciparum adhesion receptors on that membrane are poorly understood. Laser scanning confocal microscopy (LSCM) and a novel labelling and fixation method have been used to obtain high resolution immuno-fluorescent images of erythrocyte surface PfEMP1 and internal antigens which allow analysis of the accumulation of PfEMP1 on the erythrocyte membrane during asexual development.

Methods

A novel staining technique has been developed which permits distinction between erythrocyte surface PfEMP1 and intracellular PfEMP1, in parasites whose nuclear material is exceptionally well resolved. Primary antibody detection by fluorescence is carried out on the live parasitized erythrocyte. The surface labelled cells are then fixed using paraformaldehyde and permeabilized with a non-ionic detergent to permit access of antibodies to internal parasite antigens. Differentiation between surface and internal antigens is achieved using antibodies labelled with different fluorochromes and confocal microscopy

Results

Surface exposed PfEMP1 is first detectable by antibodies at the trophozoite stage of intracellular parasite development although the improved detection method indicates that there are differences between different laboratory isolates in the kinetics of accumulation of surface-exposed PfEMP1.

Conclusion

A sensitive method for labelling surface and internal PfEMP1 with up to three different fluorochromes has been developed for laser scanning confocal optical microscopy and the analysis of the developmental expression of malaria adhesion antigens.     

Ultrastructure of malaria-infected erythrocytes

Knobs, caveolae, caveola-vesicle complexes, cytoplasmic clefts, and electron-dense material are five major ultrastructural changes found in the membrane skeleton and cytoplasm of erythrocytes infected with species of primate malaria. Knobs are electron-dense, conical evaginations of the erythrocyte surface, which are believed to mediate cytoadherence and sequestration of Plasmodium falciparum-infected erythrocytes. Caveolae and caveola-vesicle complexes are flask-shaped invaginations of the membrane skeleton, which may be involved in the uptake or export of host- or parasite-derived substances. Cytoplasmic clefts are flattened or circular membranous structures found in the erythrocyte cytoplasm between the intracellular parasite and the host cell surface. The clefts are variable in length and bounded by two or more membranes. Fine, granular electron-dense material is often found on the cytoplasmic face of clefts or in amorphous packets in the erythrocyte cytoplasm. Immunocytochemistry has demonstrated that all of these ultrastructural changes are associated with the trafficking and interaction of specific malarial antigens with the host erythrocyte.     

Structural and functional characterization of Bc28.1, major erythrocyte-binding protein from Babesia canis merozoite surface

Babesiosis (formerly known as piroplasmosis) is a tick-borne disease caused by the intraerythrocytic development of protozoa parasites from the genus Babesia. Like Plasmodium falciparum, the agent of malaria, or Toxoplasma gondii, responsible for human toxoplasmosis, Babesia belongs to the Apicomplexa family. Babesia canis is the agent of the canine babesiosis in Europe. Clinical manifestations of this disease range from mild to severe and possibly lead to death by multiple organ failure. The identification and characterization of parasite surface proteins represent major goals, both for the understanding of the Apicomplexa invasion process and for the vaccine potential of such antigens. Indeed, we have already shown that Bd37, the major antigenic adhesion protein from Babesia divergens, the agent of bovine babesiosis, was able to induce complete protection against various parasite strains. The major merozoite surface antigens of Babesia canis have been described as a 28-kDa membrane protein family, anchored at the surface of the merozoite. Here, we demonstrate that Bc28.1, a major member of this multigenic family, is expressed at high levels at the surface of the merozoite. This protein is also found in the parasite in vitro culture supernatants, which are the basis of effective vaccines against canine babesiosis. We defined the erythrocyte binding function of Bc28.1 and determined its high resolution solution structure using NMR spectroscopy. Surprisingly, although these proteins are thought to play a similar role in the adhesion process, the structure of Bc28.1 from B. canis appears unrelated to the previously published structure of Bd37 from B. divergens. Site-directed mutagenesis experiments also suggest that the mechanism of the interaction with the erythrocyte membrane could be different for the two proteins. The resolution of the structure of Bc28 represents a milestone for the characterization of the parasite erythrocyte binding and its interaction with the host immune system.     

Transport of an Mr approximately 300,000 Plasmodium falciparum protein (Pf EMP 2) from the intraerythrocytic asexual parasite to the cytoplasmic face of the host cell membrane

The profound changes in the morphology, antigenicity, and functional properties of the host erythrocyte membrane induced by intraerythrocytic parasites of the human malaria Plasmodium falciparum are poorly understood at the molecular level. We have used mouse mAbs to identify a very large malarial protein (Mr approximately 300,000) that is exported from the parasite and deposited on the cytoplasmic face of the erythrocyte membrane. This protein is denoted P. falciparum erythrocyte membrane protein 2 (Pf EMP 2). The mAbs did not react with the surface of intact infected erythrocytes, nor was Pf EMP 2 accessible to exogenous proteases or lactoperoxidase-catalyzed radioiodination of intact cells. The mAbs also had no effect on in vitro cytoadherence of infected cells to the C32 amelanotic melanoma cell line. These properties distinguish Pf EMP 2 from Pf EMP 1, the cell surface malarial protein of similar size that is associated with the cytoadherent property of P. falciparum-infected erythrocytes. The mAbs did not react with Pf EMP 1. In one strain of parasite there was a significant difference in relative mobility of the 125I-surface-labeled Pf EMP 1 and the biosynthetically labeled Pf EMP 2, further distinguishing these proteins. By cryo-thin-section immunoelectron microscopy we identified organelles involved in the transit of Pf EMP through the erythrocyte cytoplasm to the internal face of the erythrocyte membrane where the protein is associated with electron-dense material under knobs. These results show that the intraerythrocytic malaria parasite has evolved a novel system for transporting malarial proteins beyond its own plasma membrane, through a vacuolar membrane and the host erythrocyte cytoplasm to the erythrocyte membrane, where they become membrane bound and presumably alter the properties of this membrane to the parasite's advantage.     

Erythrocyte and reticulocyte binding-like proteins of Plasmodium falciparum

The global agenda for malaria eradication would benefit from development of a highly efficacious vaccine that protects against disease and interrupts transmission of Plasmodium falciparum. It is likely that such a vaccine will be multi-component, with antigens from different stages of the parasite life cycle. In this review, inclusion of blood stage antigens in such a vaccine is discussed. Erythrocyte binding-like (EBL) and P. falciparum reticulocyte binding-like (PfRh) proteins are reviewed with respect to their function in erythrocyte invasion, their role in eliciting antibodies contributing to protective immunity and reduction of invasion, leading subsequently to inhibition of parasite multiplication.     

A potential novel mechanism for the insertion of a membrane protein revealed by a biochemical analysis of the Plasmodium falciparum cytoadherence molecule PfEMP-1

Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is exposed on the surface of infected erythrocytes where it both acts as an important pathogenicity factor in malaria and undergoes antigenic variation as a means of immune evasion. Because the mammalian erythrocyte lacks a protein secretory machinery there has been much interest in elucidating the mechanism whereby this protein is transferred from its site of synthesis within the parasite to its final destination. Current opinion favours a mechanism whereby PfEMP-1 becomes cotranslationally inserted into the endoplasmic reticulum of the parasite and is subsequently transported as an integral part of an erythrocyte cytoplasmic membrane system derived from the parasite. Here we show that the solubility characteristics of this protein during several stages of its transport pathway are inconsistent with this view. Instead we propose that the protein is synthesized as a peripheral membrane protein which only when it arrives at its final destination assumes a transmembrane topology. Even in this state, the extractability of the protein with urea suggest that it is anchored in the membrane by protein-protein rather than by protein-lipid interaction.     

Naturally acquired immune responses against Plasmodium falciparum sporozoites and liver infection

Malaria is a vector-borne infectious disease caused by infection with eukaryotic pathogens termed Plasmodium. Epidemiological hallmarks of Plasmodium falciparum malaria are continuous re-infections, over which time the human host may experience several clinical malaria episodes, slow acquisition of partial protection against infection, and its partial decay upon migration away from endemic regions. To overcome the exposure-dependence of naturally acquired immunity and rapidly elicit robust long-term protection are ultimate goals of malaria vaccine development. However, cellular and molecular correlates of naturally acquired immunity against either parasite infection or malarial disease remain elusive. Sero-epidemiological studies consistently suggest that acquired immunity is primarily directed against the asexual blood stages. Here, we review available data on the relationship between immune responses against the Anopheles mosquito-transmitted sporozoite and exo-erythrocytic liver stages and the incidence of malaria. We discuss current limitations and research opportunities, including the identification of additional sporozoite antigens and the use of systematic immune profiling and functional studies in longitudinal cohorts to look for pre-erythrocytic signatures of naturally acquired immunity.     

Failure to respond to the surface of Plasmodium falciparum infected erythrocytes predicts susceptibility to clinical malaria amongst African children

Following infection with Plasmodium falciparum malaria, children in endemic areas develop antibodies specific to antigens on the parasite-infected red cell surface of the infecting isolate, antibodies associated with protection against subsequent infection with that isolate. In some circumstances induction of antibodies to heterologous parasite isolates also occurs and this has been suggested as evidence for cross-reactivity of responses against the erythrocyte surface. The role of these relatively cross-reactive antibodies in protection from clinical malaria is currently unknown. We studied the incidence of clinical malaria amongst children living on the coast of Kenya through one high transmission season. By categorising individuals according to their pre-season parasite status and antibody response to the surface of erythrocytes infected with four parasite isolates we were able to identify a group of children, those who failed to make a concomitant antibody response in the presence of an asymptomatic parasitaemia, at increased susceptibility to clinical malaria in the subsequent 6 months. The fact that this susceptible group was identified regardless of the parasite isolate tested infers a cross-reactive or conserved target is present on the surface of infected erythrocytes. Identification of this target will significantly aid understanding of naturally acquired immunity to clinical malaria amongst children in endemic areas.