Antibodies and Plasmodium falciparum merozoites

There is considerable interest in using merozoite proteins in a vaccine against falciparum malaria. Observations that antibodies to merozoite surface proteins block invasion are a basis for optimism. This article draws attention to important and varied aspects of how antibodies to Plasmodium falciparum merozoites affect red blood cell invasion.     

Expression of cloned cDNA for a major surface antigen of Plasmodium falciparum merozoites

A cDNA library of P. falciparum was constructed. Using size-selected mRNA as a probe several clones were isolated which hybridized to mRNAs larger than 5 kilobases (kb). The cDNA insert of pFC 17, which hybridizes to 5.6-kb mRNA was expressed by fusion to anthranilate synthetase I in a plasmid expression vector. The expressed fusion protein was shown to contain epitopes of a 195-kDa protein which is the precursor to 3 major surface antigens of P. falciparum merozoites.     

Maurer's clefts-restricted localization, orientation and export of a Plasmodium falciparum RIFIN

RIFINs are clonally variant antigens expressed in Plasmodium falciparum. Transfection and the green fluorescence protein (GFP) tagged either internally or C-terminally to the 3D7 PFI0050c RIFIN gene product were used to investigate protein localization, orientation and trafficking. Green fluorescence pattern emerging from live transfectant parasites expressing each of the RIFIN-GFP chimera was different. The internally GFP-tagged protein was exported to Maurer's clefts (MC) in the erythrocyte cytosol, whereas the C-terminally GFP-tagged full-length RIFIN chimera was not trafficked out of the parasite. Interestingly, when some RIFIN-specific C-terminal amino acid sequences were removed, the resulting truncated molecule reached the MC. Using anti-RIFIN and anti-GFP antibodies to probe both live and fixed transfectants, staining was confined to MC and was not detected on the erythrocyte surface, a location previously suggested for this protein family. From selective permeabilization experiments, the highly variable portion of the RIFIN-GFP-insertion chimera appeared to be exposed to the erythrocyte cytosol, presumably anchored in the MC membrane via the two transmembrane domains. Trafficking of both chimeras in young ring stages was sensitive to Brefeldin A (BFA), although older rings showed differential sensitivity to BFA.     

Initial extracellular development in vitro of merozoites of Plasmodium falciparum

Late schizonts from continuous cultures of P. falciparum were concentrated over Percoll, inoculated to various experimental media at the rate of about 20 X 10(6) per 0.5 ml of medium, and incubated in a candle jar at 37 degrees for 1 day. Controls in standard culture medium showed a heavy invasion with young rings in the previously uninfected red cells introduced with the inoculum of schizonts. In a medium of high potassium content containing a 33% extract of human erythrocytes, this invasion was inhibited and many free merozoites were present. If, however, this same medium was supplemented with both ATP, as the dipotassium salt at 1.6 mM, and sodium pyruvate at 3.6 mM, there appeared large numbers of extracellular forms resembling young rings. Examination of these by electron microscopy shows that they are indeed merozoites that have begun to differentiate extracellularly. This suggests that the trigger for differentiation of merozoites may not depend on the process of entry into a red cell but rather on specific factors within the red cell.     

Characterization with monoclonal antibodies of a surface antigen of Plasmodium falciparum merozoites

The merozoite, the extracellular form of the erythrocyte stage of the malarial parasite, invades the erythrocyte and develops intracellularly. Cloned hybridoma cell lines secreting monoclonal antibodies directed against the merozoite surface were selected by indirect immunofluorescent assay by using intact isolated merozoites. Monoclonal antibodies to a 200,000 m.w. merozoite surface antigen were selected and were used to characterize this protein and its role in erythrocyte invasion. Immunoelectron microscopy demonstrated that the antigen was located exclusively on the merozoite surface coat, distributed evenly over the entire surface. The 200,000 m.w. protein incorporated [3H]glucosamine, suggesting that it is a glycoprotein and could be purified to homogeneity by using immuno-affinity chromatography. Freshly isolated, invasive merozoites retained the 200,000 m.w. antigen, but the protein was rapidly cleaved to proteins of 90,000 and 50,000 m.w. when the merozoite was extracellular. The 50,000 m.w. fragment was retained the epitope binding to monoclonal antibody 5B1 and were labeled with [3H]glucosamine. Monoclonal antibodies against the 200,000 m.w. antigen partially inhibited merozoite invasion into erythrocytes.     

Cloning and expression in Escherichia coli of a surface antigen of Plasmodium falciparum merozoites.

A complementary DNA library was constructed from mRNA purified from asexual blood forms of Plasmodium falciparum. Among the members of this library we have identified a plasmid (pMC31-1) coding for a polypeptide exposed at the surface of merozoites, the invasive stage of the asexual cycle. This plasmid was identified by direct expression using both polyclonal and monoclonal antibodies specific for a schizont polypeptide of 200 kd which has been shown to be processed to an 83-kd polypeptide expressed at the surface of merozoites. The cDNA portion of the pMC31-1 plasmid hybridizes with DNA from three isolates of P. falciparum. Antisera raised against extracts of Escherichia coli harbouring pMC31-1 react with surface and internal structures of schizonts and with the surface of merozoites from all the isolates of P. falciparum examined. These results suggest that plasmid pMC31-1 encodes an antigen of value for the development of a vaccine against malaria.     

Plasmodium falciparum Exported Protein 1 is localized to dense granules in merozoites

Apical organellar proteins in Plasmodium falciparum merozoites play important roles upon invasion. To date, dense granule, the least studied apical organelle, secretes parasite proteins across the parasitophorous vacuole membrane (PVM) to remodel the infected erythrocyte. Although this phenomenon is key to parasite growth and virulence, only five proteins so far have been identified as dense granule proteins. Further elucidation of dense granule molecule(s) is therefore required. P. falciparum Exported Protein (EXP) 1, previously reported as a parasitophorous vacuole membrane (PVM) protein, is considered essential for parasite growth. In this study, we characterized EXP1 using specific anti-EXP1 antibodies generated by immunization of wheat germ cell-free produced recombinant EXP1. Immunofluorescence microscopy (IFA) demonstrated that EXP1 co-localized with RESA, indicating that the protein is initially localized to dense granules in merozoites, followed by translocation to the PVM. The EXP1 localization in dense granule of merozoites and its translocation to the PVM after invasion of erythrocytes were further confirmed by immunoelectron microscopy. Here, we demonstrate that EXP1 is one of the dense granule proteins in merozoites, which is then transported to the PVM after invasion.     

Plasmodium falciparum: Rosettes do not protect merozoites from invasion-inhibitory antibodies

Rosetting is a parasite adhesion phenotype associated with severe malaria in African children. Why parasites form rosettes is unknown, although enhanced invasion or immune evasion have been suggested as possible functions. Previous work showed that rosetting does not enhance parasite invasion under standard in vitro conditions. We hypothesised that rosetting might promote invasion in the presence of host invasion-inhibitory antibodies, by allowing merozoites direct entry into the erythrocytes in the rosette and so minimising exposure to plasma antibodies. We therefore investigated whether rosetting influences invasion in the presence of invasion-inhibitory antibodies to MSP-1. We found no difference in invasion rates between isogenic rosetting and non-rosetting lines from two parasite strains, R29 and TM284, in the presence of MSP-1 antibodies (P = 0.62 and P = 0.63, Student's t test, TM284 and R29, respectively). These results do not support the hypothesis that rosettes protect merozoites from inhibitory antibodies during invasion. The biological function of rosetting remains unknown.     

Structural diversity of the major surface antigen of Plasmodium falciparum merozoites.

The structures of the major merozoite surface antigen of Plasmodium falciparum and the gene encoding it were indistinguishable for the Wellcome strain and the Thai clone T9/94 but different for clones T9/96, T9/98, and T9/101. The central portion of the gene is subject to the greatest variation in structure. The protein from all five lines was found to be posttranslationally modified by covalent addition of both carbohydrate and fatty acid.     

Plasmodium falciparum: isolation and purification of spontaneously released merozoites by nylon membrane sieves

A new procedure for isolating spontaneously released merozoites from in vitro cultures of Plasmodium falciparum (FVO and FCB strains) is described. The mature forms of relatively synchronous cultures containing predominantly trophozoites and few schizonts were concentrated with Plasmagel and then incubated at 37 C, without adding fresh red blood cells, until trophozoites matured into schizonts. Merozoites which were subsequently released were harvested and freed from host red blood cell material by low-speed centrifugations and nylon membrane sieves (3- and 1.2-μm pore size). From a culture containing about 5.2 × 10⁹ mature-form parasites, a total of about 10.7 × 10⁹ merozoites were released during three consecutive harvests and about 69% of these merozoites were recovered after the isolation and purification procedures. As demonstrated by both light and electron microscopy, most merozoites were morphologically intact and the merozoite preparations were free of host cell constituents. SDS-acrylamide gel electrophoresis confirmed the absence of host cell material and also showed that merozoites had a complex protein pattern of apparent molecular weights between 225 and 15 kdaltons. Such purified merozoite preparations will be invaluable for malaria immunization studies, for identification of protective antigens of P. falciparum, and for other immunological and biochemical studies.     

Antibodies targeting the PfRH1 binding domain inhibit invasion of Plasmodium falciparum merozoites

Invasion by the malaria merozoite depends on recognition of specific erythrocyte surface receptors by parasite ligands. Plasmodium falciparum uses multiple ligands, including at least two gene families, reticulocyte binding protein homologues (RBLs) and erythrocyte binding proteins/ligands (EBLs). The combination of different RBLs and EBLs expressed in a merozoite defines the invasion pathway utilized and could also play a role in parasite virulence. The binding regions of EBLs lie in a conserved cysteine-rich domain while the binding domain of RBL is still not well characterized. Here, we identify the erythrocyte binding region of the P. falciparum reticulocyte binding protein homologue 1 (PfRH1) and show that antibodies raised against the functional binding region efficiently inhibit invasion. In addition, we directly demonstrate that changes in the expression of RBLs can constitute an immune evasion mechanism of the malaria merozoite.     

Pf155/RESA antigen is localized in dense granules of Plasmodium falciparum merozoites

Immunoelectron microscopy demonstrated the presence of Pf155/RESA in dense granules of Plasmodium falciparum merozoites rather than in micronemes as previously suggested. Since the dense granules are released after the merozoite enters the parasitophorous vacuole, the role of Pf155/RESA in invasion and subsequent steps of parasite development may differ from that of a molecule located in the micronemes.     

Protein p126: a parasitophorous vacuole antigen associated with the release of Plasmodium falciparum merozoites

The p126 protein is synthesized by P. falciparum between the 32nd and the 36th hour of the erythrocytic cycle, and is localized in the parasitophorous vacuole. It is processed when schizonts rupture and the major fragments (50, 47 and 18 kDa), which are released into culture supernatant, have been characterized using monoclonal antibodies. The 47 kDa fragment has been mapped at the N-terminus of the molecule. The portion of the protein p126 gene coding for this fragment contains 3 introns and is characterized by a sequence coding for 6 repeats of 8 aminoacids and by repeats of TCA/T-AGT coding for a polyserine sequence of 37 serines in a row for the FCR-3 strain. The 50 kDa fragment is also found in culture supernatant when merozoites are released from mature schizonts. The incubation of mature schizonts with leupeptin inhibits the release of merozoites and, in this case, a 56 kDa intermediate product is found. In those conditions, merozoites were observed free in the erythrocyte cytoplasm, the membrane of the parasitophorous vacuole being destroyed. The 50 kDa fragment can be obtained from the 56 kDa fragment by treatment with trypsin (a protease inhibited by leupeptin). Our results suggest that the processing of the 56 kDa fragment: 1) is protease-dependent, and could depend on a trypsin-like activity; 2) cannot occur after the release of merozoites because of the protease inhibitors contained in the serum; 3) does not occur before the release of merozoites, since no processed products of the protein p126 are observed in unruptured schizonts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmodium falciparum merozoites: isolation by density gradient centrifugation using Percoll and antigenic analysis

Merozoites of Plasmodium falciparum were isolated and immunocytochemically analyzed. Mature parasites from knobby (K+) and knobless (K-) strains were incubated for 4 to 5 hr in RPMI 1640 with 10% serum and 10% RBC extract. About 12 to 14% of the merozoites released were recovered by density gradient centrifugation using Percoll. From 1 to 3 X 10(9) merozoites were obtained per collection. The merozoite preparations were contaminated with 10% residual bodies, about 0.1% infected and uninfected erythrocytes, about 0.1% RBC-free trophozoites and schizonts, and numerous small (less than 0.5 microns) membrane vesicles. Merozoites from the K+ and K- strains were morphologically and, by an indirect, ferritin-labeled antibody assay using serum from immune Aotus, antigenically indistinguishable. Although the residual body coats reacted with the immune Aotus serum, the membrane vesicles, some of which were seen to be blebbing from merozoites, did not react with this serum or a serum against erythrocytes. This paper describes a procedure that can be used to obtain large numbers of merozoites with little contamination by host erythrocytes.     

Electron tomography of Plasmodium falciparum merozoites reveals core cellular events that underpin erythrocyte invasion

Erythrocyte invasion by merozoites forms of the malaria parasite is a key step in the establishment of human malaria disease. To date, efforts to understand cellular events underpinning entry have been limited to insights from non‐human parasites, with no studies at sub‐micrometer resolution undertaken using the most virulent human malaria parasite, Plasmodium falciparum. This leaves our understanding of the dynamics of merozoite sub‐cellular compartments during infectionincomplete, in particular that of the secretory organelles. Using advances in P. falciparum merozoite isolation and new imaging techniques we present a three‐dimensional study of invasion using electron microscopy, cryo‐electron tomography and cryo‐X‐ray tomography. We describe the core architectural features of invasion and identify fusion between rhoptries at the commencement of invasion as a hitherto overlooked event that likely provides a critical step that initiates entry. Given the centrality of merozoite organelle proteins to vaccine development, these insights provide a mechanistic framework to understand therapeutic strategies targeted towards the cellular events of invasion.     

Cerebral malaria is associated with IgG2 and IgG4 antibody responses to recombinant Plasmodium falciparum RIFIN antigen

RIFIN proteins belong to the largest Plasmodium falciparum multicopy family of variant surface antigens (VSA) expressed by infected erythrocytes. VSA antibodies have been shown to be associated with protection against malaria. Here, antibody subclass responses to a recombinant RIFIN protein (RIF-29) in 116 Ghanaian children were determined by ELISA to investigate the relationship between severe malaria and anti-RIF-29 antibodies. The study group was composed of 23 children diagnosed exclusively for cerebral malaria and 35 children who had non-cerebral severe malaria. The remaining 58 individuals were age-, gender- and area-matched asymptomatic controls. The finding that IgG1 and IgG3 responses predominated in severe malaria patients compared to matched controls suggests that these antibodies are not protective, but are most probably induced by a current infection, an observation substantiated by the equally high reactivity to both recombinant RIF-29 protein and to P. falciparum crude lysate proteins. The exclusive detection of IgG2 and IgG4 antibodies to RIF-29 protein only in cerebral malaria children brings to mind the possibility that these antibodies are pathogenic. This is a new finding that may go some way towards explaining why these children are at risk of developing the life-threatening form of cerebral malaria.     

Extensive differential protein phosphorylation as intraerythrocytic Plasmodium falciparum schizonts develop into extracellular invasive merozoites

Pathology of the most lethal form of malaria is caused by Plasmodium falciparum asexual blood stages and initiated by merozoite invasion of erythrocytes. We present a phosphoproteome analysis of extracellular merozoites revealing 1765 unique phosphorylation sites including 785 sites not previously detected in schizonts. All MS data have been deposited in the ProteomeXchange with identifier PXD001684 ( http://proteomecentral.proteomexchange.org/dataset/PXD001684 ). The observed differential phosphorylation between extra and intraerythrocytic life‐cycle stages was confirmed using both phospho‐site and phospho‐motif specific antibodies and is consistent with the core motif [K/R]xx[pS/pT] being highly represented in merozoite phosphoproteins. Comparative bioinformatic analyses highlighted protein sets and pathways with established roles in invasion. Within the merozoite phosphoprotein interaction network a subnetwork of 119 proteins with potential roles in cellular movement and invasion was identified and suggested that it is coregulated by a further small subnetwork of protein kinase A (PKA), two calcium‐dependent protein kinases (CDPKs), a phosphatidyl inositol kinase (PI3K), and a GCN2‐like elF2‐kinase with a predicted role in translational arrest and associated changes in the ubquitinome. To test this notion experimentally, we examined the overall ubiquitination level in intracellular schizonts versus extracellular merozoites and found it highly upregulated in merozoites. We propose that alterations in the phosphoproteome and ubiquitinome reflect a starvation‐induced translational arrest as intracellular schizonts transform into extracellular merozoites.     

A co-ligand complex anchors Plasmodium falciparum merozoites to the erythrocyte invasion receptor band 3

In Plasmodium falciparum malaria, erythrocyte invasion by circulating merozoites may occur via two distinct pathways involving either a sialic acid-dependent or -independent mechanism. Earlier, we identified two nonglycosylated exofacial regions of erythrocyte band 3 termed 5ABC and 6A as an important host receptor in the sialic acid-independent invasion pathway. 5ABC, a major segment of this receptor, interacts with the 42-kDa processing product of merozoite surface protein 1 (MSP1(42)) through its 19-kDa C-terminal domain. Here, we show that two regions of merozoite surface protein 9 (MSP9), also known as acidic basic repeat antigen, interact directly with 5ABC during erythrocyte invasion by P. falciparum. Native MSP9 as well as recombinant polypeptides derived from two regions of MSP9 (MSP9/Delta1 and MSP9/Delta2) interacted with both 5ABC and intact erythrocytes. Soluble 5ABC added to the assay mixture drastically diminished the binding of MSP9 to erythrocytes. Recombinant MSP9/Delta1 and MSP9/Delta2 present in the culture medium blocked P. falciparum reinvasion into erythrocytes in vitro. Native MSP9 and MSP1(42), the two ligands binding to the 5ABC receptor, existed as a stable complex. Our results establish a novel concept wherein the merozoite exploits a specific complex of co-ligands on its surface to target a single erythrocyte receptor during invasion. This new paradigm poses a new challenge in the development of a vaccine for blood stage malaria.     

Requirement of malarial protease in the invasion of human red cells by merozoites of Plasmodium falciparum

Highly synchronous cultures of the erythrocyte stages of Plasmodium falciparum were used to determine the effects of a number of protease inhibitors on parasite development and merozoite invasion. Leupeptin, N-tosyl-L-lysyl chloromethylketone and pepstatin at a concentration greater than 0.05 mM were deleterious to both parasite development and merozoite invasion whereas aprotinin, antipain, alpha-1-antitrypsin and soybean trypsin inhibitor had no effect at a concentration of 0.5 mM. On the other hand, N-tosyl-L-phenylalanyl chloromethylketone and phenylmethylsulfonylfluoride at a concentration of 1 mM and chymostatin at a concentration of 0.15 mM inhibited merozoite invasion but were not deleterious to parasite development. Pretreatment of red cells with these three inhibitors did not block merozoite invasion. These results suggested that a chymotrypsin-like activity of the merozoite is important in the invasion process.