Erythrocytic Merozoites Research Articles

Breadth of Fc-mediated effector function correlates with clinical immunity following human malaria challenge

Malaria is a life-threatening disease of global health importance, particularly in sub-Saharan Africa. The growth inhibition assay (GIA) is routinely used to evaluate, prioritize, and quantify the efficacy of malaria blood-stage vaccine candidates but does not reliably predict either naturally acquired or vaccine-induced protection. Controlled human malaria challenge studies in semi-immune volunteers provide an unparalleled opportunity to robustly identify mechanistic correlates of protection. We leveraged this platform to undertake a head-to-head comparison of seven functional antibody assays that are relevant to immunity against the erythrocytic merozoite stage of Plasmodium falciparum. Fc-mediated effector functions were strongly associated with protection from clinical symptoms of malaria and exponential parasite multiplication, while the gold standard GIA was not. The breadth of Fc-mediated effector function discriminated clinical immunity following the challenge. These findings present a shift in the understanding of the mechanisms that underpin immunity to malaria and have important implications for vaccine development.

Histone modification analysis reveals common regulators of gene expression in liver and blood stage merozoites of Plasmodium parasites

Gene expression in malaria parasites is subject to various layers of regulation, including histone post-translational modifications (PTMs). Gene regulatory mechanisms have been extensively studied during the main developmental stages of Plasmodium parasites inside erythrocytes, from the ring stage following invasion to the schizont stage leading up to egress. However, gene regulation in merozoites that mediate the transition from one host cell to the next is an understudied area of parasite biology. Here, we sought to characterize gene expression and the corresponding histone PTM landscape during this stage of the parasite lifecycle through RNA-seq and ChIP-seq on P. falciparum blood stage schizonts, merozoites, and rings, as well as P. berghei liver stage merozoites. In both hepatic and erythrocytic merozoites, we identified a subset of genes with a unique histone PTM profile characterized by a region of H3K4me3 depletion in their promoter. These genes were upregulated in hepatic and erythrocytic merozoites and rings, had roles in protein export, translation, and host cell remodeling, and shared a DNA motif. These results indicate that similar regulatory mechanisms may underlie merozoite formation in the liver and blood stages. We also observed that H3K4me2 was deposited in gene bodies of gene families encoding variant surface antigens in erythrocytic merozoites, which may facilitate switching of gene expression between different members of these families. Finally, H3K18me and H2K27me were uncoupled from gene expression and were enriched around the centromeres in erythrocytic schizonts and merozoites, suggesting potential roles in the maintenance of chromosomal organization during schizogony. Together, our results demonstrate that extensive changes in gene expression and histone landscape occur during the schizont-to-ring transition to facilitate productive erythrocyte infection. The dynamic remodeling of the transcriptional program in hepatic and erythrocytic merozoites makes this stage attractive as a target for novel anti-malarial drugs that may have activity against both the liver and blood stages.

A new modeling and existence–uniqueness analysis for Babesiosis disease of fractional order

In this study, we consider the dynamics of the Babesiosis transmission on bovine populations and ticks. The most important role in the transmission of the parasite is the ticks from the Ixodidae family. The vector tick takes factors (merozoites in erythrocytes) from the diseased animal while sucking the blood. To model and investigate the transmissions of this parasite and address this important issue, we have considered the disease in a fractional epidemiological model. This paper, therefore, discusses the mechanisms of transmission of Babesiosis defined in the fractional derivative sense. The Caputo–Fabrizio (CF) derivative is considered to study the propagation mechanisms of Babesiosis. First, the important characteristics of the model have been presented, and then the transmission of the Babesiosis model defined in CF is discussed. The application of fixed-point theory is used to derive the concept of the qualitative properties of the mentioned model. The solution is obtained by using the Homotopy perturbation Elzaki transform method (HPETM). Numerical simulations are performed, and the effects of the arbitrary-order derivatives are investigated graphically.

A Chimeric Plasmodium vivax Merozoite Surface Protein Antibody Recognizes and Blocks Erythrocytic P. cynomolgi Berok Merozoites In Vitro.

Research on erythrocytic Plasmodium vivax merozoite antigens is critical for identifying potential vaccine candidates in reducing P. vivax disease. However, many P. vivax studies are constrained by its inability to undergo long-term culture in vitro Conserved across all Plasmodium spp., merozoite surface proteins are essential for invasion into erythrocytes and highly expressed on erythrocytic merozoites, thus making it an ideal vaccine candidate. In clinical trials, the P. vivax merozoite surface protein 1 (PvMSP1-19) vaccine candidate alone has shown to have limited immunogenicity in patients; hence, we incorporate the highly conserved and immunogenic C terminus of both P. vivax merozoite surface protein 8 (PvMSP8) and PvMSP1-19 to develop a multicomponent chimeric protein rPvMSP8+1 for immunization of mice. The resulted chimeric rPvMSP8+1 antibody was shown to recognize native protein MSP8 and MSP1-19 of mature P. vivax schizonts. In the immunized mice, an elevated antibody response was observed in the rPvMSP8+1-immunized group compared to that immunized with single-antigen components. In addition, we examined the growth inhibition of these antibodies against Plasmodium cynomolgi (Berok strain) parasites, which is phylogenetically close to P. vivax and sustains long-term culture in vitro Similarly, the chimeric anti-rPvMSP8+1 antibodies recognize P. cynomolgi MSP8 and MSP1-19 on mature schizonts and showed strong inhibition in vitro via growth inhibition assay. This study provides support for a new multiantigen-based paradigm rPvMSP8+1 to explore potential chimeric vaccine candidates against P. vivax malaria using sister species P. cynomolgi.

Deletion of the rodent malaria ortholog for falcipain-1 highlights differences between hepatic and blood stage merozoites.

Proteases have been implicated in a variety of developmental processes during the malaria parasite lifecycle. In particular, invasion and egress of the parasite from the infected hepatocyte and erythrocyte, critically depend on protease activity. Although falcipain-1 was the first cysteine protease to be characterized in P. falciparum, its role in the lifecycle of the parasite has been the subject of some controversy. While an inhibitor of falcipain-1 blocked erythrocyte invasion by merozoites, two independent studies showed that falcipain-1 disruption did not affect growth of blood stage parasites. To shed light on the role of this protease over the entire Plasmodium lifecycle, we disrupted berghepain-1, its ortholog in the rodent parasite P. berghei. We found that this mutant parasite displays a pronounced delay in blood stage infection after inoculation of sporozoites. Experiments designed to pinpoint the defect of berghepain-1 knockout parasites found that it was not due to alterations in gliding motility, hepatocyte invasion or liver stage development and that injection of berghepain-1 knockout merosomes replicated the phenotype of delayed blood stage growth after sporozoite inoculation. We identified an additional role for berghepain-1 in preparing blood stage merozoites for infection of erythrocytes and observed that berghepain-1 knockout parasites exhibit a reticulocyte restriction, suggesting that berghepain-1 activity broadens the erythrocyte repertoire of the parasite. The lack of berghepain-1 expression resulted in a greater reduction in erythrocyte infectivity in hepatocyte-derived merozoites than it did in erythrocyte-derived merozoites. These observations indicate a role for berghepain-1 in processing ligands important for merozoite infectivity and provide evidence supporting the notion that hepatic and erythrocytic merozoites, though structurally similar, are not identical.

Plasmodium delichoni n. sp.: description, molecular characterisation and remarks on the exoerythrocytic merogony, persistence, vectors and transmission.

Malaria parasite Plasmodium (Novyella) delichoni n. sp. (Haemosporida, Plasmodiidae) was found in a widespread Eurasian songbird, the common house martin Delichon urbicum (Hirundinidae). It is described based on the morphology of its blood stages and segments of the mitochondrial cytochrome b and apicoplast genes, which can be used for molecular identification of this species. Erythrocytic meronts and gametocytes are strictly nucleophilic, and mature gametocytes possess pigment granules of markedly variable size, including large ones (1μm in length). Due to these features, P. delichoni can be readily distinguished from all described species of avian malaria parasites belonging to subgenus Novyella. Additionally, mature erythrocytic merozoites contain a dense clump of chromatin, a rare character in avian malaria parasites. Erythrocytic merogony is asynchronous. Illustrations of blood stages of the new species are given, and phylogenetic analysis identifies DNA lineages closely related to this parasite. Domestic canary Serinus canaria and Eurasian siskin Carduelis spinus were infected after subinoculation of infected blood obtained from the house martin. Parasitemia was long lasting in both these hosts, but it was high (up to 70%) in Eurasian siskins and low (up to 1%) in canaries. Mortality was not observed, and histological examination and chromogenic in situ hybridisation did not reveal secondary exoerythrocytic meronts (phanerozoites) in the exposed birds. It is likely that persistence of this infection occurs due to long-lasting parasitemia in avian hosts. Sporogony was abortive in mosquitoes Culex pipiens pipiens form molestus, Culex quinquefasciatus and Aedes aegypti at gametogenesis or ookinete stages. The new species is absent from juvenile birds at breeding sites in Europe, indicating that transmission occurs at African wintering grounds.

Optimal control of malaria chemotherapy

We present an intra-host mathematical model of malaria that describes the interaction of the immune system with the blood stage malaria merozoites. The model is modified by incorporating the effects of malaria drugs that target blood stage parasites. The optimal control represents a percentage effect of the chemotherapy of chloroquine in combination with chlorpheniramine on the reproduction of merozoites in erythrocytes. First we maximise the benefit based on the immune cells, and minimise the systemic cost based on the percentage of chemotherapies given and the population of merozoites. An objective functional to minimise merozite reproduction and treatment systemic costs is then built. The existence and uniqueness results for the optimal control are established. The optimality system is derived and the Runge–Kutta fourth order scheme is used to numerically simulate different therapy efforts. Our results indicate that highly toxic drugs with the compensation of high infection suppression have the potential of yeilding better treatment results than less toxic drugs with less infection suppression potential or high toxic drugs with less infection suppression potential. In addition, we also observed that a treatment protocol with drugs with high adverse effects and with a high potential of merozoite suppression can be beneficial to patients. However, an optimal control strategy that seeks to maximise immune cells has no potential to improve the treatment of blood stage malaria.

O-GlcNAc modification of the anti-malarial vaccine candidate PfAMA1: in silico-defined structural changes and potential to generate a better vaccine

The complex life cycle of plasmodial parasites makes the selection of a single subunit protein a less than optimal strategy to generate an efficient vaccinal protection against malaria. Moreover, the full protection afforded by malarial proteins carried by intact parasites implies that immune responses against different antigens expressed in different phases of the cycle are required, but also suggests that native malarial antigens are presented to the host immune system in a manner that recombinant proteins do not achieve. The malarial apical membrane antigen 1 (AMA1) represents a suitable vaccine candidate because AMA1 is expressed on sporozoites and merozoites and allows them to invade hepatocytes and erythrocytes, respectively. Anti-AMA1 antibodies and cytotoxic T-cells are therefore expected to interfere both with the primary invasion of hepatocytes by sporozoites and with the later propagation of merozoites in erythrocytes, and thus efficiently counteract parasite development in its human host. AMA1 bears potential glycosylation sites and the human erythrocytic O-linked N-acetylglucosamine transferase (OGT) could glycosylate AMA1 through combinatorial metabolism. This hypothesis was tested in silico by developing binding models of AMA1 with human OGT complexed with UDP-GlcNc, and followed by the binding of O-GlcNAc with the hydroxyl group of AMA1 serine and threonine residues. Our results suggests that AMA1 shows potential for glycosylation at Thr517 and Ser498 and that O-GlcNAc AMA1 may constitute a conformationally more appropriate antigen for developing a protective anti-malarial immune response.

Plasmodium falciparum Inhibitory Capacities of Paired Maternal-cord Sera from South-west Province, Cameroon

In malaria endemic areas, young children are protected against malaria attack during the first few weeks of life partially by transplacentally acquired antibodies. In this study, we show, using an in vitro assay, that part of these antibodies are involved with blocking the re-invasion of host red blood cells by erythrocytic merozoites. One hundred consecutive paired maternal-cord blood samples were collected at delivery and their plasma assayed for total IgG antibodies against crude blood stage antigens by the ELISA. The Ig fraction were precipitated from the plasma samples with (NH(4))(2)SO(4), purified on PD10 columns and used in vitro in determining the re-invasion inhibitory capacities. The mean (+/-SD) ELISA OD(405) IgG antibodies to crude blood stage antigens of maternal (0.476 +/- 0.48) and cord (0.421 +/- 0.39) plasma samples was not significantly different. However, the mean total protein concentration of the Ig fractions for maternal samples (15.82 +/- 3.85) was significantly higher (p=0.005) than that of paired cord samples (12.87 +/- 2.86 mg/ml). There was no correlation between anti-Plasmodium falciparum-specific IgG levels and total protein concentrations of the Ig fractions of both maternal and cord samples. The entire test Ig fractions were strongly inhibitory (>50 per cent) except for four paired maternal cord samples, which were moderately inhibitory (21--50 per cent) at the highest concentration tested (1:2 dilution). Furthermore, there was no correlation between maternal IgG levels and percentage re-invasion inhibition at the 1:2 dilution. The results suggest that mothers resident in malaria endemic areas possess naturally acquired re-invasion inhibitory antibodies and their foetuses can acquire these antibodies transplacentally, which may contribute to the relative protection observed in infants during their first few weeks of life.

Production of monoclonal antibodies against a 19-kD recombinant Plasmodium vivax MSP1 for detection of P. vivax malaria in Turkey.

Plasmodium vivax malaria, which is transmitted to humans by mosquitoes, is one of the most important parasitic diseases in Turkey. The major protein on the surface of asexual erythrocytic stage merozoites of P. vivax (Pv) is 200 kD and called major merozoite surface protein-1 (PvMSP1). Polyclonal antibodies against the 19-kD C-terminal fragment of PvMSP1 (PvMSP1(19)) are protective in monkey models of P. vivax and associated with protection in field studies. In this research, monoclonal antibodies were produced against PvMSP1(19). A total of 214 IgG(1) antibody-releasing hybridomas were obtained and three monoclonal antibodies were produced (PvMSP1(19).1, PvMSP1(19).2, and PvMSP1(19).3) and selected for further study. They have now been purified from ascitic fluid on a Staphylococcus protein A affinity column. These are the first monoclonal antibodies produced against P. vivax in Turkey and the first monoclonal antibodies produced against this recombinant PvMSP1(19) in the world. The monoclonal antibodies will be used to study the epidemiology of P. vivax in patients with malaria in Turkey, and to develop better strategies for early diagnosis and treatment of the disease in our population.

Stage-specific transcription of distinct repertoires of a multigene family during Plasmodium life cycle.

Members of a multigene family in the rodent malaria parasite Plasmodium yoelii yoelii code for 235-kilodalton proteins (Py235) that are located in the merozoite apical complex, are implicated in virulence, and may determine red blood cell specificity. We show that distinct subsets of py235 genes are expressed in sporozoites and hepatic and erythrocytic stages. Antibodies to Py235 inhibited sporozoite invasion of hepatocytes. The switch in expression profile occurred immediately after transition from one stage to another. The results suggest that this differential expression is driven by strong biological requirements and provide evidence that hepatic and erythrocytic merozoites differ.

Vaccination against tropical theileriosis.

Theileria annulata, the cause of tropical theileriosis is propagated in cattle with stage-to-stage transmission by Hyalomma ticks. Three stages in the life cycle of the parasite--tick-derived sporozoites, intramononuclear schizonts, and erythrocytic merozoites--infect cattle. When cattle are inoculated with schizont-infected cells, the parasite is transferred from the donor cell to the recipient. The main pathological damage in cattle is induced by the schizont stage. Each development stage of T. annulata elicits a specific immune response. Schizont-infected lymphoid cells can be grown indefinitely in culture and prolonged cultivation results in loss of virulence. Blood-derived schizonts induce stronger immunity than culture-derived schizonts, which suggests that restrictions on the parasite population or antigenic variation occur during prolonged cultivation. The duration of immunity following sporozoite or schizont infections has not yet been determined, but does not appear to be lifelong. The attenuated, culture-derived anti-theileria vaccine proved to be safe and effective in prevention of field theileriosis in large enzootic areas.

The Life History, Ultrastructure, and Experimental Transmission of Hepatozoon catesbianae n. comb., an Apicomplexan Parasite of the Bullfrog, Rana catesbeiana and the Mosquito, Culex territans in Algonquin Park, Ontario

Gametogenesis and sporogonic development of a haemogregarine parasite of bullfrogs (Rana catesbeiana) was observed in cells of the Malpighian tubules of laboratory-reared Culex territans that had fed on naturally infected bullfrogs. Mature oocysts, which varied considerably in size, were multisporocystic with ellipsoidal sporocysts that contained 4 sporozoites. Sporogonic development was completed in about 20 days. Mature meronts were observed in the liver and merozoites in erythrocytes of laboratory-reared bullfrogs that had been fed sporocysts 19 days previously. Similar attempts to infect laboratory-reared green and northern leopard frogs experimentally were unsuccessful, suggesting rather narrow specificity for this parasite in ranids. Gametogenesis and sporogonic stages of this parasite were ultrastructurally similar to those described for Hepatozoon species. The parasite appears to be transmitted directly between bullfrogs and mosquitoes in the study area where Cx. territans feeds avidly on bullfrogs, which in turn were observed to naturally ingest these mosquitoes. Based on data presented in this study and the earlier description by Stebbins in 1903, the haemogregarine parasite of bullfrogs was designated as a new combination, Hepatozoon catesbianae.

Expression of Plasmodium Berghei Circumsporozoite Antigen on the Surface of Exoerythrocytic Schizonts and Merozoites

The intracellular distribution of circumsporozoite (CS) antigen was traced by immunoelectron microscopy in cultures of Plasmodium berghei exoerythrocytic (EE) schizonts with monoclonal antibody (Mab) 3D11 to the immunodominant repeat region of the P. berghei CS protein. CS antigen was localized on the parasitophorous vacuole (PV) membrane and pellicular complex of recently invaded sporozoites and on electron-dense masses of sloughed CS antigen in the host cell cytoplasm. CS antigen persisted throughout the complete EE cycle of P. berghei on the surface of EE schizonts and was incorporated into the plasma membrane of budding EE merozoites. Erythrocytic merozoites were not labeled by Mab 3D11, indicating that these 2 populations of merozoites differ in antigenic composition. Significant internal labeling occurred in 50 hr EE schizonts in association with the limiting membranes of peripheral vesicles and short, tube-like structures attached to their outer surfaces. These vesicles contained an electron-dense flocculent material also present in the PV space. Association of CS antigen with the limiting membranes of these vesicles suggests that they either develop as endocytotic invaginations of the schizont plasma membrane or transport newly synthesized CS antigen from the endoplasmic reticulum and Golgi of developing EE schizonts to the parasite surface.

Freeze fracture study of the schizonts and merozoites of Leucocytozoon caulleryi

The fine structure of the schizonts and merozoites of Leucocytozoon caulleryi was studied by the freeze fracture technique. Young schizonts were spherical, surrounded by a thick fibrous capsule and possessed numerous nuclei. The nucleus was surrounded by the outer and inner nuclear membrane. A number of nuclear pores were evident in the nuclear membranes. Advanced schizonts were characterized by the formation of cytomeres. Each cytomere was surrounded by a membrane. Several nuclei lay along the periphery of the cytomere. The protoplasmic face (P-face) of the membrane of the cytomere contained numerous intramembranous particles (IMP). The exoerythrocytic merozoites were surrounded by a two-layered pellicle consisting of the outer and inner membrane. During freeze fracturing, each of the two membranes could split along its hydrophobic interior to yield a total of four fracture faces. The P-faces of the outer and inner membranes were closely covered with evenly distributed IMP. The external faces (E-face) of these membranes had less IMP than the P-faces. The membrane architecture of the erythrocytic merozoites was similar to that of the exoerythrocytic merozoites. The erythrocytic merozoite was enveloped by an additional membrane probably derived from the plasma membrane of host cell.

Structure and development of the surface coat of erythrocytic merozoites of Plasmodium knowlesi.

The surface of extracellular merozoites of P. knowlesi is covered with a coat 15-20 nm thick, made up of clusters of filaments standing erect on the plasma membrane. Filaments have stems 2 nm thick, the peripheral ends of which are complex, branching or ending in long trailing threads. Coat filaments occur on the surface of the parasite in regular rows at an early schizont stage, and persist until well after merozoite release. They are sensitive to trypsin and papain, and bind ethanolic phosphotungstate, indicating a proteinaceous nature. They are also removed by exposure to phosphate-buffered saline. Filaments bear negative charges, binding cationised ferritin throughout the depth of the coat and staining with ruthenium red. They cover the whole merozoite surface and mediate intercellular adhesion at distances of 15-150 nm, membrane to membrane. It is suggested that these filaments correspond to a major merozoite surface protein, and are important in the initial capture of red cells.

Lamellar membranes associated with rhoptries in erythrocytic merozoites ofPlasmodium knowlesi: a clue to the mechanism of invasion

In merozoites of Plasmodium knowlesi, rhoptries have a dense substructure of fine (2.5 nm diameter) granules and short rods. These are not altered by lipid extraction, and stain with ethanolic phosphotungstate indicating a proteinaceous composition. Various types of fixation also show multilamellar whorls with a periodicity of 5-7 nm in the tips of rhoptries or extruded at the merozoite apex. In merozoites fixed during invasions of red cells, membrane continuity typically occurs between the rim of the rhoptry canal and the red cell membrane, but where this contact has apparently been lost, extensive membranous whorls and blebs are often found at the apex of the parasite. Similar structures occur at the apices of merozoites within late-stage schizonts. It is suggested that the same mechanism which generates these lamellae forms the parasitophorous vacuole by inserting membranous elements formed by the parasite into the red cell membrane, so causing its invagination. A similar mechanism may be responsible for the release of merozoites from the late-stage schizont.

Development of Erythrocytic Merozoites to Gametocytes in Chickens Recovered from Sporozoite Infection with Leucocytozoon caulleryi

Development of Erythrocytic Merozoites to Gametocytes in Chickens Recovered from Sporozoite Infection with Leucocytozoon caulleryi

Ultrastructural localization of protective antigens of Plasmodium yoelii merozoites by the use of monoclonal antibodies and ultrathin cryomicrotomy.

The production of two hybridoma cell lines secreting monoclonal antibodies (MAb), both of which react specifically with erythrocytic merozoites of Plasmodium yoelii in the indirect immunofluorescence assay, has been reported earlier. MAb 25.77 was reactive with a localized region within each merozoite, while MAb 25.1 appeared to be specific for the plasma membrane of schizonts and merozoites. The parasite antigens recognized by antibodies 25.77 and 25.1 are proteins of 235,000 and 230,000 molecular weight, respectively, both of which induce protective immunity against P. yoelii in mice. In order to establish the precise localization of these protective antigens within erythrocyte merozoites, ultrathin cryomicrotomy was used in conjunction with the MAb and protein A-gold. This technique showed that gold particles were exclusively concentrated over the rhoptries when erythrocytic merozoites were incubated with MAb 25.77. On the other hand, gold particles were distributed uniformly over the merozoite surface when parasites were incubated with MAb 25.1. These results demonstrate, for the first time, that a protective antigen of the erythrocytic stage of P. yoelii is localized within the rhoptries as well as on the merozoite surface.

The chemotherapy of rodent malaria, XXXVII

Two new Mannich base antimalarials, WR 194,965 and 228,258 exhibit blood schizontocidal action against Plasmodium berghei in vivo. This action is retained also against the P. berghei NS line which has a low level of resistance to chloroquine. While WR 228,258 is also active against the highly chloroquine-resistant RC line, WR 194,965 is not. Both compounds show slightly reduced activity against the highly mefloquine-resistant N/1100 line. WR 225,448, an 8-aminoquinoline, is as active against blood stages of the NS, RC and N/1100 lines of P. berghei as against the N strain, and more so on a dose/weight basis than the first two compounds. It shows a low level of cross-resistance against the primaquine-resistant P line. WR 225,448 has a clear causal prophylactic effect on the pre-erythrocytic schizonts of P. yoelii nigeriensis. While WR 194,965 and 228,258 also appeared to act on these stages, the effect is most probably due to residual action on emerging erythrocytic merozoites since pre-erythrocytic schizonts in treated animals were morphologically indistinguishable from those of the untreated control.