Localization and function of a Plasmodium falciparum protein (PF3D7_1459400) during erythrocyte invasion.

IMPACT STATEMENT: Plasmodium falciparum malaria is a global health problem. Erythrocyte invasion by P. falciparum merozoites appears to be a promising target to curb malaria. We have identified and characterized a novel protein that is involved in erythrocyte invasion. Our data on protein subcellular localization, stage-specific protein expression pattern, and merozoite invasion inhibition by α-peptide antibodies suggest a role for PF3D7_1459400 protein during P. falciparum erythrocyte invasion. Even more, the human immunoepidemiology data present PF3D7_1459400 protein as an immunogenic antigen which could be further exploited for the development of new anti-infective therapy against malaria.

Molecular Characterization and Immuno-Reactivity Patterns of a Novel Plasmodium falciparum Armadillo-Type Repeat Protein, PfATRP.

Nearly half of the genes in the Plasmodium falciparum genome have not yet been functionally investigated. We used homology-based structural modeling to identify multiple copies of Armadillo repeats within one uncharacterized gene expressed during the intraerythrocytic stages, PF3D7_0410600, subsequently referred to as P. falciparum Armadillo-Type Repeat Protein (PfATRP). Soluble recombinant PfATRP was expressed in a bacterial expression system, purified to apparent homogeneity and the identity of the recombinant PfATRP was confirmed by mass spectrometry. Affinity-purified α-PfATRP rabbit antibodies specifically recognized the recombinant protein. Immunofluorescence assays revealed that α-PfATRP rabbit antibodies reacted with P. falciparum schizonts. Anti-PfATRP antibody exhibited peripheral staining patterns around the merozoites. Given the localization of PfATRP in merozoites, we tested for an egress phenotype during schizont arrest assays and demonstrated that native PfATRP is inaccessible on the surface of merozoites in intact schizonts. Dual immunofluorescence assays with markers for the inner membrane complex (IMC) and microtubules suggest partial colocalization in both asexual and sexual stage parasites. Using the soluble recombinant PfATRP in a screen of plasma samples revealed that malaria-infected children have naturally acquired PfATRP-specific antibodies.

Antibody Reactivity to Merozoite Antigens in Ghanaian Adults Correlates With Growth Inhibitory Activity Against Plasmodium falciparum in Culture.

BACKGROUND: Plasmodium falciparum uses a repertoire of merozoite-stage proteins for invasion of erythrocytes. Antibodies against some of these proteins halt the replication cycle of the parasite by preventing erythrocyte invasion and are implicated as contributors to protective immunity against malaria. METHODS: We assayed antibody reactivity against a panel of 9 recombinant antigens based on erythrocyte-binding antigen (EBA) and reticulocyte-like homolog (Rh) proteins in plasma from children with malaria and healthy adults residing in 3 endemic areas in Ghana using enzyme-linked immunosorbent assay. Purified immunoglobulin (Ig)G from adult plasma samples was also tested for invasion inhibition against 7 different P falciparum culture lines, including clinical isolates. RESULTS: Antibodies against the antigens increased in an age-dependent manner in children. Breadth of reactivity to the different antigens was strongly associated with in vitro parasite growth inhibitory activity of IgG purified from the adults. The strongest predictors of breadth of antibody reactivity were age and transmission intensity, and a combination of reactivities to Rh2, Rh4, and Rh5 correlated strongly with invasion inhibition. CONCLUSIONS: Growth inhibitory activity was significantly associated with breadth of antibody reactivity to merozoite antigens, encouraging the prospect of a multicomponent blood-stage vaccine.

Functional Characterization of Plasmodium falciparum Surface-Related Antigen as a Potential Blood-Stage Vaccine Target.

Plasmodium falciparum erythrocyte invasion is a multistep process that involves a spectrum of interactions that are not well characterized. We have characterized a 113-kDa immunogenic protein, PF3D7_1431400 (PF14_0293), that possesses coiled-coil structures. The protein is localized on the surfaces of both merozoites and gametocytes, hence the name Plasmodium falciparum surface-related antigen (PfSRA). The processed 32-kDa fragment of PfSRA binds normal human erythrocytes with different sensitivities to enzyme treatments. Temporal imaging from initial attachment to internalization of viable merozoites revealed that a fragment of PfSRA, along with PfMSP119, is internalized after invasion. Moreover, parasite growth inhibition assays showed that PfSRA P1 antibodies potently inhibited erythrocyte invasion of both sialic acid-dependent and -independent parasite strains. Also, immunoepidemiological studies show that malaria-infected populations have naturally acquired antibodies against PfSRA. Overall, the results demonstrate that PfSRA has the structural and functional characteristics of a very promising target for vaccine development.

Assessing the impact of differences in malaria transmission intensity on clinical and haematological indices in children with malaria.

BACKGROUND: Malaria control interventions have led to a decline in transmission intensity in many endemic areas, and resulted in elimination in some areas. This decline, however, will lead to delayed acquisition of protective immunity and thus impact disease manifestation and outcomes. Therefore, the variation in clinical and haematological parameters in children with malaria was assessed across three areas in Ghana with varying transmission intensities. METHODS: A total of 568 children between the ages of 2 and 14 years with confirmed malaria were recruited in hospitals in three areas with varying transmission intensities (Kintampo > Navrongo > Accra) and a comprehensive analysis of parasitological, clinical, haematological and socio-economic parameters was performed. RESULTS: Areas of lower malaria transmission tended to have lower disease severity in children with malaria, characterized by lower parasitaemias and higher haemoglobin levels. In addition, total white cell counts and percent lymphocytes decreased with decreasing transmission intensity. The heterozygous sickle haemoglobin genotype was protective against disease severity in Kintampo (P = 0.016), although this was not significant in Accra and Navrongo. Parasitaemia levels were not a significant predictor of haemoglobin level after controlling for age and gender. However, higher haemoglobin levels in children were associated with certain socioeconomic factors, such as having fathers who had any type of employment (P < 0.05) and mothers who were teachers (P < 0.05). CONCLUSIONS: The findings demonstrate significant differences in the haematological presentation and severity of malaria among areas with different transmission intensity in Ghana, indicating that these factors need to be considered in planning the management of the disease as the endemicity is expected to decline after control interventions.

Variation in Plasmodium falciparum erythrocyte invasion phenotypes and merozoite ligand gene expression across different populations in areas of malaria endemicity.

Plasmodium falciparum merozoites use diverse alternative erythrocyte receptors for invasion and variably express cognate ligands encoded by the erythrocyte binding antigen (eba) and reticulocyte binding-like homologue (Rh) gene families. Previous analyses conducted on parasites from single populations in areas of endemicity revealed a wide spectrum of invasion phenotypes and expression profiles, although comparisons across studies have been limited by the use of different protocols. For direct comparisons within and among populations, clinical isolates from three different West African sites of endemicity (in Ghana, Guinea, and Senegal) were cryopreserved and cultured ex vivo after thawing in a single laboratory to assay invasion of target erythrocytes pretreated with enzymes affecting receptor subsets. Complete invasion assay data from 67 isolates showed no differences among the populations in the broad range of phenotypes measured by neuraminidase treatment (overall mean, 40.6% inhibition) or trypsin treatment (overall mean, 83.3% inhibition). The effects of chymotrypsin treatment (overall mean, 79.2% inhibition) showed heterogeneity across populations (Kruskall-Wallis P = 0.023), although the full phenotypic range was seen in each. Schizont-stage transcript data for a panel of 8 invasion ligand genes (eba175, eba140, eba181, Rh1, Rh2a, Rh2b, Rh4, and Rh5) were obtained for 37 isolates, showing similar ranges of variation in each population except that eba175 levels tended to be higher in parasites from Ghana than in those from Senegal (whereas levels of eba181 and Rh2b were lower in parasites from Ghana). The broad diversity in invasion phenotypes and gene expression seen within each local population, with minimal differences among them, is consistent with a hypothesis of immune selection maintaining parasite variation.

Analysis of Erythrocyte Invasion Mechanisms of Plasmodium falciparum Clinical Isolates Across 3 Malaria-Endemic Areas in Ghana.

BACKGROUND: Plasmodium falciparum invades human erythrocytes by using an array of ligands that interact with several receptors, including sialic acid (SA), complement receptor 1 (CR1), and basigin. We hypothesized that in malaria-endemic areas, parasites vary invasion pathways under immune pressure. Therefore, invasion mechanisms of clinical isolates collected from 3 zones of Ghana with different levels of endemicity (from lowest to highest, Accra, Navrongo, and Kintampo) were compared using standardized methods. METHODS: Blood samples were collected from children aged 2-14 years in whom malaria was diagnosed, and erythrocyte invasion phenotypes were determined using the enzymes neuraminidase, chymotrypsin, and trypsin, which differentially cleave receptors from the erythrocyte surface. In addition, antibodies against CR1 and basigin were used to determine the contributions of these receptors to invasion. Gene expression levels of P. falciparum invasion ligands were also examined. RESULTS: The parasites generally expressed SA-independent invasion phenotypes across the malaria-endemic areas, with parasites from Kintampo showing the highest invasion rates in neuraminidase-treated erythrocytes. CR1 was a major mediator of SA-independent invasion, while basigin was essential for both SA-dependent and SA-independent invasion mechanisms. Furthermore, expression of the basigin ligand PfRh5 was the best predictor of donor parasitemia. CONCLUSIONS: Erythrocyte invasion phenotypes expressed by P. falciparum are influenced by endemicity levels, and the PfRh5-basigin pathway is a potential vaccine target.

Age-related pattern and monocyte-acquired haemozoin associated production of erythropoietin in children with severe malarial anaemia in Ghana.

BACKGROUND: Malaria continues to be a global health challenge, affecting more than half the world's population and causing approximately 660,000 deaths annually. The majority of malaria cases are caused by Plasmodium falciparum and occur in sub-Saharan Africa. One of the major complications asscociated with malaria is severe anaemia, caused by a cycle of haemoglobin digestion by the parasite. Anaemia due to falciparum malaria in children has multifactorial pathogenesis, which includes suppression of bone marrow activity. Recent studies have shown that haemozoin, which is a by-product of parasite haemoglobin digestion, may play an important role in suppression of haemoglobin production, leading to anaemia. In this study we correlated the levels of erythropoietin (EPO), as an indicator of stimulation of haemoglobin production, to the levels of monocyte acquired haemozoin in children with both severe and uncomplicated malaria. There was a significantly negative correlation between levels of haemozoin-containing monocytes and EPO, which may suggest that haemozoin suppresses erythropoiesis in severe malaria. A multiple linear regression analysis and simple bar was used to investigate associations between various haematological parameters. METHODS: To examine the levels of erythropoietin in the age categories, the levels of erythropoietin was measured using a commercial Enyme-Linked Immunosorbent Assay (ELISA). Giemsa-stained blood smears were used to determine percentage pigment containing monocytes. The haemozoin containing monocytes was expressed as a percentage of the total number of monocytes. To obtain the number of haemozoin containing monocytes/µL the percentage of haemozoin containing monocytes was multiplied by the absolute number of monocytes/µL from the automated haematology analyzer. RESULTS: The levels of erythropoietin in younger children (<3 years) was significantly higher than in older children with a similar degree of malaria anaemia (Hb levels) (p < 0.005). Haemozoin-containing monocytes were relatively higher in severe malaria anaemia patients compared to those with uncomplicated malaria (p < 0.001). CONCLUSIONS: Age purportedly has a direct effect on background levels of erythropoietin. With corresponding decreased levels of erythropoietin in older children with the same degree of severe malarial anaemia, conceivably, the bone marrows of younger children with acute malaria may be less sensitive to erythropoietin.