Regioselectively modified sulfated cellulose as prospective drug for treatment of malaria tropica

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to placental chondroitin-4-sulfate (CSA) has been linked to the severe disease outcome of pregnancy-associated malaria. Consequently, sulfated polysaccharides with inhibitory capacity may be considered for therapeutic strategies as anti-adhesive drugs. During in vitro screening a regioselectively modified cellulose sulfate (CS10) was selected as prime candidate for further investigations because it was able to inhibit adhesion to CSA expressed on CHO cells and placental tissue, to de-adhere already bound infected erythrocytes, and to bind to infected erythrocytes. Similar to the undersulfated placental CSA preferred by placental-binding infected erythrocytes, CS10 is characterized by a clustered sulfate pattern along the polymer chain. In further evaluation of its effects on P. falciparum interactions with host erythrocytes, we now show that CS10 inhibits the in vitro asexual growth of parasites in erythrocytes. Furthermore, we show that CS10 interferes with C1 of the classical complement pathway but not with MBL of the lectin pathway. In order to gain insights into the possible interactions of CS10 with known parasite receptors at the molecular level, we designed 3D-structures of characteristic stretches of CS10. CS10 fragments with clustered sulfate groups showed complex patterns of hydrophobic and hydrophilic patches most likely suitable for interactions with protein binding partners. The significance of CS10 interactions with the complement system as well as its anti-malarial effect for prospective drug application are discussed.     

Chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of Plasmodium falciparum infected erythrocytes.

BACKGROUND: Chondroitin-4-sulfate (CSA) was recently described as a Plasmodium falciparum cytoadherence receptor present on Saimiri brain microvascular and human lung endothelial cells. MATERIALS AND METHODS: To specifically study chondroitin-4-sulfate-mediated cytoadherence, a parasite population was selected through panning of the Palo-Alto (FUP) 1 P. falciparum isolate on monolayers of Saimiri brain microvascular endothelial cells (SBEC). Immunofluorescence showed this SBEC cell line to be unique for its expression of CSA-proteoglycans, namely CD44 and thrombomodulin, in the absence of CD36 and ICAM-1. RESULTS: The selected parasite population was used to monitor cytoadherence inhibition/dissociating activities in Saimiri sera collected at different times after intramuscular injection of 50 mg CSA/kg of body weight. Serum inhibitory activity was detectable 30 min after injection and persisted for 8 hr. Furthermore, when chondroitin-4-sulfate was injected into monkeys infected with Palo-Alto (FUP) 1 P. falciparum, erythrocytes containing P. falciparum mature forms were released into the circulation. The cytoadherence phenotype of circulating infected red blood cells (IRBC) was determined before and 8 hr after inoculation of CSA. Before inoculation, in vitro cytoadherence of IRBCs was not inhibited by CSA. In contrast, in vitro cytoadherence of circulating infected erythrocytes obtained 8 hr after CSA inoculation was inhibited by more than 90% by CSA. CONCLUSIONS: In the squirrel monkey model for infection with P. falciparum, chondroitin-4-sulfate impairs in vitro and in vivo cytoadherence of parasitized erythrocytes.     

The human placental derived BeWo cell line: a useful model for selecting Plasmodium falciparum CSA-binding parasites

Chondroitin sulfate A (CSA) present in the placental intervillous blood spaces has been described as the main receptor involved in the massive sequestration of Plasmodium falciparum parasitized erythrocytes to the placenta. Placental parasite isolates are functionally distinct from isolates that sequester in other organs, because they do not cytoadhere to CD36 but instead bind to CSA. To investigate for the parasites molecules associated with the CSA adhesion phenotype, different methodologies have been developed to select for CSA-binding lines in vitro mainly using non-placental sources of CSA that differ in their sulfation pattern. In this study, we show that the human trophoblastic BeWo cell line is a very efficient alternative to select for the CSA-binding phenotype in parasitized erythrocytes.     

Identification of a 67-amino-acid region of the Plasmodium falciparum variant surface antigen that binds chondroitin sulphate A and elicits antibodies reactive with the surface of placental isolates

The complications of malaria in pregnancy are caused by the massive sequestration of parasitized erythrocytes (PE) in the placenta. Placental isolates of Plasmodium falciparum are unusual in that they do not bind the primary microvasculature receptor CD36 but instead bind chondroitin sulphate A (CSA). Pregnant mothers develop antibodies that recognize placental variants worldwide, suggesting that a vaccine against malaria in pregnancy is possible. Some members of the Duffy binding-like gamma (DBL-gamma) domain of the large and diverse P. falciparum erythrocyte membrane protein-1 (PfEMP-1) family, when expressed on Chinese hamster ovary (CHO) cells, bind CSA. To characterize better the molecular requirements for DBL-gamma adhesion to CSA, we determined the binding of various DBL-gamma domains. Most DBL-gamma did not bind CSA, and no conserved region was identified that strictly differentiated binders from non-binders. Structure-function analysis of the FCR3-CSA DBL-gamma domain localized the minimal CSA binding region to a 67-residue fragment. This region was partially conserved among some binding sequences. Serum from a rabbit immunized with the minimal domain reacted with CSA-binding parasite lines, but not with non-CSA-adherent PE lines that adhered to CD36 and other receptors. The identification of a minimal binding region from a highly variable cytoadherent family may have application for a vaccine against malaria in pregnancy.     

Plasmodium falciparum: cloned and expressed CIDR domains of PfEMP1 bind to chondroitin sulfate A

Adherence of erythrocytes infected with mature asexual Plasmodium falciparum parasites (iRBC) to microvascular endothelial cells contributes to the pathology of P. falciparum malaria. It has been shown that the variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) confers adhesion to a wide range of cell surface receptors. Previously, the cysteine-rich interdomain region (CIDR) of PfEMP1 has been identified as binding site to CD36. We provide evidence that the same region can also mediate binding to chondroitin sulfate A (CSA). CIDR domains of two different parasite strains were expressed in Escherichia coli as a 6xHis-tagged protein. Purified recombinant protein bound to Chinese hamster ovary (CHO) cells which naturally express chondroitin sulfate A. Treatment of wild-type CHO cells with chondroitinase ABC reduced binding up to 94.4%. Competitive binding using soluble CSA inhibited binding to CHO cells by up to 100% at 2 mg/ml and by 62.4% at 0.5 mg/ml, whereas 1 mg/ml heparan sulfate had only a little effect (18.1%). In contrast, a recombinant 6xHis-tagged DBL1 domain showed no binding to wild-type CHO cells. Such an approach of analyzing various domains of PfEMP1 as recombinant proteins may elucidate their functions and may lead to novel anti-adherence therapeutics, especially for maternal malaria infections.     

CD36 directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes

Erythrocytes infected with P. falciparum express knob-like adhesion structures that allow the infected cells to cling to the postcapilliary endothelium of characteristic host organs. At present, the mechanism of cytoadherence is not fully understood. While parasitized erythrocytes have been shown to specifically bind to the platelet/matrix molecule thrombospondin, adherence to suitable target cells can also be blocked by monoclonal antibody OKM5, which recognizes a surface molecule expressed by hematopoietic cells and endothelium. In apparent reconciliation of these findings, it has been reported that the OKM5 antigen (CD36) is a receptor for thrombospondin. Here we report that expression of a CD36 cDNA clone in COS cells supports cytoadherence of parasitized erythrocytes but does not support increased binding of purified human thrombospondin.     

Adhesion of Plasmodium falciparum-infected erythrocytes to hyaluronic acid in placental malaria

Infection with Plasmodium falciparum during pregnancy leads to the accumulation of parasite-infected erythrocytes in the placenta, and is associated with excess perinatal mortality, premature delivery and intrauterine growth retardation in the infant, as well as increased maternal mortality and morbidity. P. falciparum can adhere to specific receptors on host cells, an important virulence factor enabling parasites to accumulate in various organs. We report here that most P. falciparum isolates from infected placentae can bind to hyaluronic acid, a newly discovered receptor for parasite adhesion that is present on the placental lining. In laboratory isolates selected for specific high-level adhesion, binding to hyaluronic acid could be inhibited by dodecamer or larger oligosaccharide fragments or polysaccharides, treatment of immobilized receptor with hyaluronidase, or treatment of infected erythrocytes with trypsin. In vitro flow-based assays demonstrated that high levels of adhesion occurred at low wall shear stress, conditions thought to prevail in the placenta. Our findings indicate that adhesion to hyaluronic acid is involved in mediating placental parasite accumulation, thus changing the present understanding of the mechanisms of placental infection, with implications for the development of therapeutic and preventative interventions.     

Investigation of host factors possibly enhancing the emergence of the chondroitin sulfate A-binding phenotype in Plasmodium falciparum

In malaria endemic areas, regardless of immunity acquired during lifelong exposure to malaria, pregnant women become susceptible to Plasmodium falciparum infections. Malaria during pregnancy is associated with a massive sequestration of infected erythrocytes in the placenta and the emergence of a unique parasite-derived adhesive molecule (encoded by var2CSA) that binds to chondroitin sulfate A (CSA). How P. falciparum achieves the timely expression of the CSA ligand in pregnant women remains puzzling. We investigated whether host serum-specific factors present only during pregnancy may induce var2CSA expression. Our panel of experiments did not reveal significant changes in var2CSA levels and CSA-binding capacity.     

A single member of the Plasmodium falciparum var multigene family determines cytoadhesion to the placental receptor chondroitin sulphate A

In high-transmission regions, protective clinical immunity to Plasmodium falciparum develops during the early years of life, limiting serious complications of malaria in young children. Pregnant women are an exception and are especially susceptible to severe P. falciparum infections resulting from the massive adhesion of parasitized erythrocytes to chondroitin sulphate A (CSA) present on placental syncytiotrophoblasts. Epidemiological studies strongly support the feasibility of an intervention strategy to protect pregnant women from disease. However, different parasite molecules have been associated with adhesion to CSA. In this work, we show that disruption of the var2csa gene of P. falciparum results in the inability of parasites to recover the CSA-binding phenotype. This gene is a member of the var multigene family and was previously shown to be composed of domains that mediate binding to CSA. Our results show the central role of var2CSA in CSA adhesion and support var2CSA as a leading vaccine candidate aimed at protecting pregnant women and their fetuses.     

Evaluating specific adhesion of Plasmodium falciparum-infected erythrocytes to immobilised hyaluronic acid with comparison to binding of mammalian cells

A feature of infection with Plasmodium falciparum is the ability of parasite-infected erythrocytes to adhere to vascular endothelial cells and accumulate in vital organs, associated with severe clinical disease. Hyaluronic acid was recently identified as a receptor for adhesion and has been implicated in mediating the accumulation of parasites in the placenta. Here, we report in vitro assays to measure specific adhesion of infected erythrocytes to hyaluronic acid that is distinct from binding to chondroitin sulphate A, another glycosaminoglycan implicated as a receptor in placental malaria. In this study, specific adhesion of mature stage infected erythrocytes to hyaluronic acid of high purity immobilised on plastic surfaces was abolished by pre-treating hyaluronic acid with a specific hyaluronate lyase from Streptomyces, whereas the same treatment of chondroitin sulphate A had little effect. Adhesion to hyaluronic acid could not be explained by the presence of chondroitin sulphate A or other glycosaminoglycans in the hyaluronic acid preparations. Chinese hamster ovary cells bound in a similar manner in the assays and confirmed that hyaluronic acid was appropriately immobilised for cell adhesion. In contrast to parasites, these cells did not adhere to chondroitin sulphate A. The adsorption of hyaluronic acid onto plastic surfaces was also confirmed by the use of a specific hyaluronic acid-binding protein. Fixing cells with glutaraldehyde at the completion of adhesion assays reduced the number of parasites remaining adherent to hyaluronic acid, but not to chondroitin sulphate A or CD36. These findings have important implications for understanding and evaluating interactions between P. falciparum and hyaluronic acid that may be involved in disease pathogenesis.     

Plasmodium falciparum-infected erythrocytes bind ICAM-1 at a site distinct from LFA-1, Mac-1, and human rhinovirus.

The attachment of erythrocytes infected with P. falciparum to human venular endothelium is the primary step leading to complications from severe and cerebral malaria. Intercellular adhesion molecule-1 (ICAM-1, CD54) has been implicated as a cytoadhesion receptor for P. falciparum-infected erythrocytes. Characterization of domain deletion, human/murine chimeric ICAM-1 molecules, and amino acid substitution mutants localized the primary binding site for parasitized erythrocytes to the first amino-terminal immunoglobulin-like domain of ICAM-1. The ICAM-1 binding site is distinct from those recognized by LFA-1, Mac-1, and the human major-type rhinoviruses. Synthetic peptides encompassing the binding site on ICAM-1 inhibited malaria-infected erythrocyte adhesion to ICAM-1-coated surfaces with a Ki of 0.1-0.3 mM, whereas the Ki for soluble ICAM-1 is 0.15 microM. These findings have implications for the therapeutic reversal of malaria-infected erythrocyte sequestration in the host microvasculature.     

Plasmodium falciparum: chondroitin sulfate A is the major receptor for adhesion of parasitized erythrocytes in the placenta

Plasmodium falciparum parasites that sequester in the placenta bind to the molecule chondroitin sulfate A (CSA). Women become resistant to malaria during pregnancy as they acquire antibodies that inhibit parasite adhesion to CSA, suggesting that a vaccine against placental malaria is feasible. Hyaluronic acid (HA) and non-immune IgG have also been proposed as receptors for P. falciparum adhesion in the placenta, but evidence for their roles is inconclusive. In this study, CSA, HA, and IgG were simultaneously assessed for their relative contributions to placental adhesion. Placental parasites collected in Tanzania uniformly adhered to the molecule CSA, and soluble CSA completely inhibited adhesion of most samples to placental cryosections. Three of 46 placental parasite samples also adhered to immobilized HA, but HA failed to inhibit adhesion of any placental parasites to placental cryosections. Similarly, non-immune IgG and protein A failed to inhibit adhesion of parasite samples to placental cryosection. P. falciparum adhesion in the placenta appears to be a non-redundant process that requires CSA as a receptor. Vaccines that elicit functional antibodies against CSA-binding parasites may confer resistance to pregnancy malaria.     

Microfluidic modeling of cell-cell interactions in malaria pathogenesis

The clinical outcomes of human infections by Plasmodium falciparum remain highly unpredictable. A complete understanding of the complex interactions between host cells and the parasite will require in vitro experimental models that simultaneously capture diverse host-parasite interactions relevant to pathogenesis. Here we show that advanced microfluidic devices concurrently model (a) adhesion of infected red blood cells to host cell ligands, (b) rheological responses to changing dimensions of capillaries with shapes and sizes similar to small blood vessels, and (c) phagocytosis of infected erythrocytes by macrophages. All of this is accomplished under physiologically relevant flow conditions for up to 20 h. Using select examples, we demonstrate how this enabling technology can be applied in novel, integrated ways to dissect interactions between host cell ligands and parasitized erythrocytes in synthetic capillaries. The devices are cheap and portable and require small sample volumes; thus, they have the potential to be widely used in research laboratories and at field sites with access to fresh patient samples.     

The role of Plasmodium falciparum var genes in malaria in pregnancy

Sequestration of Plasmodium falciparum-infected erythrocytes in the placenta is responsible for many of the harmful effects of malaria during pregnancy. Sequestration occurs as a result of parasite adhesion molecules expressed on the surface of infected erythrocytes binding to host receptors in the placenta such as chondroitin sulphate A (CSA). Identification of the parasite ligand(s) responsible for placental adhesion could lead to the development of a vaccine to induce antibodies to prevent placental sequestration. Such a vaccine would reduce the maternal anaemia and infant deaths that are associated with malaria in pregnancy. Current research indicates that the parasite ligands mediating placental adhesion may be members of the P. falciparum variant surface antigen family PfEMP1, encoded by var genes. Two relatively well-conserved subfamilies of var genes have been implicated in placental adhesion, however, their role remains controversial. This review examines the evidence for and against the involvement of var genes in placental adhesion, and considers whether the most appropriate vaccine candidates have yet been identified.     

Recovery of adhesion to chondroitin-4-sulphate in Plasmodium falciparum varCSA disruption mutants by antigenically similar PfEMP1 variants

Protection against maternal malaria has been associated with the acquisition of a specific antibody response that prevents adhesion of Plasmodium falciparum-infected erythrocytes to the glycosaminoglycan chondroitin-4-sulphate (CSA), which is present in the placental intervillous space. These antibodies are directed against variant forms of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) that mediate binding to CSA. We have generated insertional disruption mutants of the gene encoding the CSA-binding phenotype in the P. falciparum clone FCR3 (varCSA) to test the hypothesis that strategies targeting the parasite's determinant for this adhesive phenotype may prevent sequestration of infected erythrocytes in the placenta and hence the development of maternal malaria. The varCSA-disruption mutants were initially unable to adhere to CSA; however, they could recover the phenotype after repeated selection over CSA. We show that recovery of CSA binding is varCSA independent and mediated by the activation of a novel var variant. Importantly, the corresponding PfEMP1 protein reacts with a monoclonal antibody recognizing the DBL3 gamma domain of the varCSA gene product, indicating that the DBL3 gamma CSA-binding domains are conserved between these PfEMP1-binding variants. Our data support strategies exploring these conserved epitopes as vaccine candidates against maternal malaria.     

Disruption of var2csa gene impairs placental malaria associated adhesion phenotype

Infection with Plasmodium falciparum during pregnancy is one of the major causes of malaria related morbidity and mortality in newborn and mothers. The complications of pregnancy-associated malaria result mainly from massive adhesion of Plasmodium falciparum-infected erythrocytes (IE) to chondroitin sulfate A (CSA) present in the placental intervillous blood spaces. Var2CSA, a member of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family is the predominant parasite ligand mediating CSA binding. However, experimental evidence suggests that other host receptors, such as hyaluronic acid (HA) and the neonatal Fc receptor, may also support placental binding. Here we used parasites in which var2csa was genetically disrupted to evaluate the contribution of these receptors to placental sequestration and to identify additional adhesion receptors that may be involved in pregnancy-associated malaria. By comparison to the wild-type parasites, the FCR3delta var2csa mutants could not be selected for HA adhesion, indicating that var2csa is not only essential for IE cytoadhesion to the placental receptor CSA, but also to HA. However, further studies using different pure sources of HA revealed that the previously observed binding results from CSA contamination in the bovine vitreous humor HA preparation. To identify CSA-independent placental interactions, FCR3delta var2csa mutant parasites were selected for adhesion to the human placental trophoblastic BeWo cell line. BeWo selected parasites revealed a multi-phenotypic adhesion population expressing multiple var genes. However, these parasites did not cytoadhere specifically to the syncytiotrophoblast lining of placental cryosections and were not recognized by sera from malaria-exposed women in a parity dependent manner, indicating that the surface molecules present on the surface of the BeWo selected population are not specifically expressed during the course of pregnancy-associated malaria. Taken together, these results demonstrate that the placental malaria associated phenotype can not be restored in FCR3delta var2csa mutant parasites and highlight the key role of var2CSA in pregnancy malaria pathogenesis and for vaccine development.     

Cytoadhesion and falciparum malaria: going with the flow

Sequestration of parasitized red blood cells in the cerebral vasculature is the predisposing event to the development of cerebral malaria during infection with Plasmodium falciparum. The adhesive interaction between these cells and receptors on the endothelial cell (cytoadhesion) occurs in the dynamic environment of the microcirculation, but most studies have neglected this factor and have concentrated on measuring adhesion in static (no flow) assays. Such studies ignore the markedly different rheological properties of parasitized red blood cells that become apparent when adhesion is examined under dynamic, flow conditions that resemble those of the circulation in vivo. Here, Brian Cooke and Ross Coppel review a number of novel aspects of cytoadhesion that have been identified using flow-based assays, and discuss their relevance to the pathophysiology, investigation and clinical management of falciparum malaria.     

Characterization of anti-var2CSA-PfEMP1 cytoadhesion inhibitory mouse monoclonal antibodies

Pregnancy-associated malaria (PAM) is associated with the massive sequestration of erythrocytes infected with CSA-binding parasites in the placenta. Natural protective immunity against PAM is acquired during the course of pregnancies, with the development of anti-PfEMP1 antibodies recognizing placental infected erythrocytes (IEs) from different geographical regions. Mouse monoclonal antibodies (mabs) were raised against Plasmodium falciparum variant surface proteins expressed by CSA-binding parasites. These mabs blocked 0-60% of CSA-binding parasite adhesion and immunoprecipitated a 350 kDa 125I-labeled PfEMP1(CSA). Two var2CSA domains expressed on the surface of CHO cells (DBL5epsilon and DBL6epsilon) were identified as the targets of three of four antibodies inhibiting CSA binding. Two of these antibodies also recognized either DBL2x or DBL3x, suggesting that some epitopes may be common to several var2CSA domains. These mabs also specifically selected CSA-binding IEs and facilitated the purification from IE extracts of the native var2CSA ligand. This purified ligand elicited antibodies in immunized mice inhibiting efficiently IE(CSA) cytoadhesion. Based on our findings, we provide the first demonstration that the parasite var2CSA surface protein can elicit inhibitory antibodies and define here the subunits of the var2CSA ligand suitable for use in vaccine development.     

Chondroitin sulphate A as an adherence receptor for Plasmodium falciparum-infected erythrocytes

Until recently, the sequestration of erythrocytes infected with Plasmodium falciparum has been thought to be due to one of a number of protein-protein interactions. In this article, Stephen Rogerson and Graham Brown summarize the emerging evidence that, in vitro, infected erythrocytes can also adhere to the glycosaminoglycan chondroitin sulphate A (CSA) expressed on the surface of cells and immobilized on plastic. In vivo, binding of infected erythrocytes to CSA could be crucial to the development of malarial infection of the placenta, and possibly to sequestration in the lung and brain. The consequences of this may include maternal morbidity and mortality, low birth weight in the infant, pulmonary oedema and cerebral malaria. They discuss the need to characterize the molecular basis of this interaction, and to investigate the possible therapeutic role of CSA in malaria. Chondroitin sulphates are nontoxic compounds already in use for other diseases in humans. Vaccines based on inhibiting this receptor-ligand interaction could also be appropriate.