Abnormal flow adhesion of sickle red blood cells to human placental trophoblast extracellular matrix.

Pregnancy in sickle cell disease (SCD) has been associated with increased complications such as vaso-occlusive crises, severe anemia and foetal loss. It has been proposed that the sickling of red blood cells (RBCs) inside the placenta circulation could participate to these complications. The present study investigated the adhesion of sickle RBCs on human trophoblast-derived cell and its extracellular matrix. Results demonstrated 1) similar adhesion of sickle RBCs and healthy RBCs to trophoblast but 2) a greater adhesion of sickle RBCs to the extracellular matrix of trophoblasts as compared with healthy RBCs. This greater adhesion could partly involve the Lu/BCAM glycoproteins and could participate to the complications reported in SCD pregnant women.

Strain-transcendent immune response to recombinant Var2CSA DBL5-ε domain block P. falciparum adhesion to placenta-derived BeWo cells under flow conditions.

BACKGROUND: Pregnancy-associated malaria (PAM) is a serious consequence of the adhesion to the placental receptor chondroitin sulfate A (CSA) of Plasmodium falciparum-infected erythrocytes (PE) expressing the large cysteine-rich multi-domain protein var2CSA. Women become resistant to PAM, and develop strain-transcending immunity against CSA-binding parasites. The identification of var2CSA regions that could elicit broadly neutralizing and adhesion-blocking antibodies is a key step for the design of prophylactic vaccine strategies. METHODOLOGY: Escherichia coli expressed var2CSA DBL domains were refolded and purified prior to immunization of mice and a goat. Protein-G-purified antibodies were tested for their ability to block FCR3(CSA)-infected erythrocytes binding to placental (BeWo) and monkey brain endothelial (ScC2) cell lines using a flow cytoadhesion inhibition assay mimicking closely the physiological conditions present in the placenta at shear stress of 0.05 Pa. DBL5-ε, DBL6-ε and DBL5-6-ε induced cross-reactive antibodies using Alum and Freund as adjuvants, which blocked cytoadhesion at values ranging between 40 to 96% at 0.5 mg IgG per ml. Importantly, antibodies raised against recombinant DBL5-ε from 3 distinct parasites genotypes (HB3, Dd2 and 7G8) showed strain-transcending inhibition ranging from 38 to 64% for the heterologuous FCR3(CSA). CONCLUSIONS: Using single and double DBL domains from var2CSA and Alum as adjuvant, we identified recombinant subunits inducing an immune response in experimental animals which is able to block efficiently parasite adhesion in a flow cytoadhesion assay that mimics closely the erythrocyte flow in the placenta. These subunits show promising features for inclusion into a vaccine aiming to protect against PAM.

Mechanisms of malarial anaemia: potential involvement of the Plasmodium falciparum low molecular weight rhoptry-associated proteins.

Plasmodium falciparum malaria is a major cause of morbidity and mortality throughout the tropics. Anaemia is a constant feature of the disease. Pregnant women mostly primigravidae and children below the age of 5 years are the most afflicted. Its pathogenesis is multifactorial and incompletely understood. Among several factors, the destruction of erythrocytes (RBCs) is the most frequently observed cause of severe malarial anaemia and the removal of non-parasitized RBCs (nEs) is thought to be the most important, accounting for approximately 90% of the reduction in haematocrit in acute malaria. Previous studies demonstrated that the tagging of nEs with the parasite antigen RAP-2 (rhoptry-associated protein-2; also designated RSP-2) due to either failed or aborted invasion by merozoites resulted in the destruction of these cells. In this study we further investigated the mechanisms mediating the destruction of nEs in the development of severe malarial anaemia and the possible involvement of RAP-2/RSP-2 and other members of the low molecular weight rhoptry complex (RAP-1: rhoptry-associated protein-1 and RAP-3: rhoptry-associated protein-3). Antibodies to the rhoptry-associated proteins were found to recognise the surface of nEs in a parasitaemia-dependent manner after merozoite release in P. falciparumin vitro cultures. These cells, as well as erythroblasts co-cultured with infected RBCs (IEs), could then be destroyed by either phagocytosis or lysis after complement activation. The ability of anti-rhoptry antibodies to mediate the destruction of RAP-2/RSP-2-tagged erythroblasts in the presence of effector cells was also investigated. Data obtained suggest that mouse monoclonal antibodies to the low molecular weight RAP proteins mediate the death of RAP-2/RSP-2-tagged erythroblasts on interaction with adherent monocytes. The mechanism of cell death is not yet fully known, but seems to involve primarily apoptosis. The above observations suggest that the antibody response against RAP-2/RSP-2 and other members of the complex could trigger the destruction of RAP-2/RSP-2-tagged host cells. Taken together it appears that during severe anaemia a defective bone marrow or dyserythropoiesis possibly due to erythroblast cell death, may overlap with the accelerated destruction of normal erythroid cells, either by opsonisation or complement activation further aggravating the anaemia which may become fatal. These observations could therefore have implications in the design, development and deployment of future therapeutic interventions against malaria.

Var2CSA DBL6-epsilon domain expressed in HEK293 induces limited cross-reactive and blocking antibodies to CSA binding parasites.

BACKGROUND: Pregnancy-associated malaria (PAM) is a serious consequence of Plasmodium falciparum-infected erythrocytes sequestration in the placenta through the adhesion to the placental receptor chondroitin sulfate A (CSA). Although women become resistant to PAM as they acquire transcending inhibitory immunity against CSA-binding parasites, hundreds of thousands of lives could be saved if a prophylactic vaccine targeting the surface proteins of placental parasites could be designed. Recent works point to the variant protein var2CSA as the key target for the development of a pregnancy-associated malaria vaccine. However, designing such a prophylactic vaccine has been hindered by the difficulty in identifying regions of var2CSA that could elicit broadly neutralizing and adhesion-blocking antibodies. METHODS: Var2CSA is a very large protein with an estimated molecular weight of 350 kDa, and can be divided into six cysteine rich Duffy binding-like domains (DBL). The human embryonic kidney 293 cell line (HEK293) was used to produce secreted soluble recombinant forms of var2CSA DBL domains. The Escherichia coli expression system was also assessed for the domains not expressed or expressed in low amount in the HEK293 system. To investigate whether var2CSA binding DBL domains can induce biologically active antibodies recognizing the native var2CSA and blocking the interaction, mice were immunized with the refolded DBL3-X or the HEK293 secreted DBL6-epsilon domains. RESULTS: Using the HEK293 expression system, DBL1-X, DBL4-epsilon and DBL6-epsilon were produced at relatively high levels in the culture supernatant, while DBL3-X and DBL5-epsilon were produced at much lower levels. DBL2-X and DBL3-X domains were obtained after refolding of the inclusion bodies produced in E. coli. Importantly, mice antisera raised against the recombinant DBL6-epsilon domain, specifically reacted against the surface of CSA-binding parasites and revealed adhesion blocking activity. CONCLUSION: This is the first report showing inhibitory binding antibodies obtained through a var2CSA recombinant DBL domain immunization protocol. These results support the current strategies using var2CSA as immunogen in the aim of blocking placental sequestration of malaria parasites. This work is a step towards the development of a var2CSA based vaccine that will prevent pregnancy-associated malaria and improve pregnancy outcomes.

CD44, a signal receptor for the inhibition of the cytoadhesion of CD36-binding Plasmodium falciparum-infected erythrocytes by CSA-binding infected erythrocytes.

The cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) in organ microvessels is a key event in the pathogenesis of cerebral malaria and pulmonary edema. Identification of the molecules involved in the interaction between IEs and endothelial cells has been a major goal of research into severe forms of malaria. In contrast, the consequences of cytoadhesion for endothelial cells have been largely ignored. By combining phenotypic selection, cytoadhesion assays and flow cytometry, we demonstrated that the cytoadhesion of CSA-binding IEs inhibited the cytoadhesion of CD36-binding IEs. We identified CD44 as a signal receptor for CSA-binding IEs cytoadhesion, and demonstrated that the signal was transduced to CD36 through a pathway involving the Src-kinase family and MEK. CD36-mediated cytoadhesion was modulated independently of changes in CD36 expression. These results provide the first evidence that some IEs can downregulate the cytoadhesion of IEs of another phenotype, by modifying endothelial cells via a signaling pathway relating CD44 to CD36. Mimicking this phenomenon may constitute an interesting therapeutic strategy for inhibiting the adhesion of CD36-binding IEs -- the most abundant phenotype among field isolates -- and promoting their degradation in the spleen.

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.

Neural cell adhesion molecule, a new cytoadhesion receptor for Plasmodium falciparum-infected erythrocytes capable of aggregation.

The cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) to the endothelial cells lining the microvasculature, clogging the microvessels of various organs, is a key event in the pathogenesis of certain severe forms of malaria, such as cerebral malaria and pulmonary edema. Studies aiming to identify possible correlations between the severity of clinical cases and the presence of particular cytoadhesion phenotypes have been largely unsuccessful. One of the possible reasons for this failure is that some of the key receptors and/or mechanisms involved have yet to be identified. By combining IE selection, cell transfection, and adhesion inhibition assays, we identified a new cytoadhesion receptor, neural cell adhesion molecule (NCAM). NCAM is a member of the immunoglobulin superfamily and has nonpolysialylated and polysialylated isoforms, the latter being rare in adults. The nonpolysialylated form is present on the surfaces of endothelial cells in the microvessels of various organs in which IE sequestration occurs. We found that multiphenotypic IEs interacted with nonpolysialylated NCAM and with another, as yet unidentified receptor. These IEs also displayed cytoadhesion in flow conditions, presenting the unique ability to form adherent macroaggregates composed of hundreds of IEs. These features may act as virulence factors, increasing the capacity of IEs to clog microvessels via receptor synergy and macroaggregate formation, thereby facilitating the pathogenesis of severe forms of malaria.

Dissection of the role of PfEMP1 and ICAM-1 in the sensing of Plasmodium-falciparum-infected erythrocytes by natural killer cells.

BACKGROUND: Host innate immunity contributes to malaria clinical outcome by providing protective inflammatory cytokines such as interferon-gamma, and by shaping the adaptive immune response. Plasmodium falciparum (Pf) is the etiologic agent of the most severe forms of human malaria. Natural Killer (NK) cells are lymphocytes of the innate immune system that are the first effectors to produce interferon-gamma in response to Pf. However, the molecular bases of Pf-NK cell recognition events are unknown. Our study focuses on the role of Pf erythrocyte membrane protein 1 (PfEMP1), a major Pf virulence factor. PfEMP1 is expressed on parasitized-erythrocytes and participates to vascular obstruction through the binding to several host receptors. PfEMP1 is also a pivotal target for host antibody response to Pf infection. METHODOLOGY/PRINCIPAL FINDINGS: Using genetically-engineered parasite mutant strains, a human genetic deficiency, and blocking antibodies, we identified two receptor-ligand pairs involved in two uncoupled events occurring during the sensing of Pf infection by NK cells. First, PfEMP1 interaction with one of its host receptor, chondroitin sulfate A, mediates the cytoadhesion of Pf-infected erythrocytes to human NK cell lines, but is not required for primary NK cell activation. Second, intercellular adhesion molecule-1 (ICAM-1), another host receptor for PfEMP1, is mandatory for NK cell interferon-gamma response. In this case, ICAM-1 acts via its engagement with its host ligand, LFA-1, and not with PfEMP1, consistent with the obligatory cross-talk of NK cells with macrophages for their production of interferon-gamma. CONCLUSION/SIGNIFICANCE: PfEMP1-independent but ICAM-1/LFA-1-dependent events occurring during NK cell activation by Pf highlight the fundamental role of cellular cooperation during innate immune response to malaria.

Pregnancy-associated malaria: parasite binding, natural immunity and vaccine development.

Humans living in areas of high malaria transmission gradually acquire, during the early years of life, protective clinical immunity to Plasmodium falciparum, limiting serious complications of malaria to young children. However, pregnant women become more susceptible to severe P. falciparum infections during their first pregnancy. Pregnancy associated malaria is coupled with massive accumulation of parasitised erythrocytes and monocytes in the placental intervillous blood spaces, contributing to disease and death in pregnant women and developing infants. Indirect evidence suggests that prevention may be possible by vaccinating women of childbearing age before their first pregnancy. This review aims to introduce the reader to the implications of malaria infection during pregnancy and to analyse recent findings towards the identification and characterisation of parasite encoded erythrocyte surface proteins expressed in malaria-infected pregnant women that are likely targets of protective immunity and have potential for vaccine development.

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.

Immunogenicity of Duffy binding-like domains that bind chondroitin sulfate A and protection against pregnancy-associated malaria.

Sequestration of Plasmodium falciparum-infected erythrocytes in the placenta is implicated in pathological outcomes of pregnancy-associated malaria (PAM). P. falciparum isolates that sequester in the placenta primarily bind chondroitin sulfate A (CSA). Following exposure to malaria during pregnancy, women in areas of endemicity develop immunity, and so multigravid women are less susceptible to PAM than primigravidae. Protective immunity to PAM is associated with the development of antibodies that recognize diverse CSA-binding, placental P. falciparum isolates. The epitopes recognized by such protective antibodies have not been identified but are likely to lie in conserved Duffy binding-like (DBL) domains, encoded by var genes, that bind CSA. Immunization of mice with the CSA-binding DBL3gamma domain encoded by var1CSA elicits cross-reactive antibodies that recognize diverse CSA-binding P. falciparum isolates and block their binding to placental cryosections under flow. However, CSA-binding isolates primarily express var2CSA, which does not encode any DBLgamma domains. Here, we demonstrate that antibodies raised against DBL3gamma encoded by var1CSA cross-react with one of the CSA-binding domains, DBL3X, encoded by var2CSA. This explains the paradoxical observation made here and earlier that anti-rDBL3gamma sera recognize CSA-binding isolates and provides evidence for the presence of conserved, cross-reactive epitopes in diverse CSA-binding DBL domains. Such cross-reactive epitopes within CSA-binding DBL domains can form the basis for a vaccine that provides protection against PAM.

Natural killer cell and macrophage cooperation in MyD88-dependent innate responses to Plasmodium falciparum.

IFN-gamma secretion by natural killer (NK) cells is pivotal to several tumor and viral immune responses, during which NK and dendritic cells cooperation is required. We show here that macrophages are mandatory for NK cell IFN-gamma secretion in response to erythrocytes infected with Plasmodium falciparum (Pf), a causative agent of human malaria. In addition, direct sensing of Pf infection by NK cells induces their production of the proinflammatory chemokine CXCL8, without triggering their granule-mediated cytolytic programs. Despite their reported role in Pf recognition, Toll-like receptor (TLR) 2, TLR9, and TLR11 are individually dispensable for NK cell activation induced by Pf-infected erythrocytes. However, IL-18R expression on NK cells, IL-18 production by macrophages, and MyD88 on both cell types are essential components of this previously undescribed pathway of NK cell activation in response to a parasite infection.

Plasmodium falciparum rhoptry protein RSP2 triggers destruction of the erythroid lineage.

The destruction of erythrocytes and defects in erythropoiesis are among the most frequently observed causes of morbidity in severe Plasmodium falciparum malaria. The molecular mechanisms involved remain unclear, despite extensive investigation. We show here, for the first time, that tagging with the parasite rhoptry protein ring surface protein 2 (RSP2) is not restricted to the surfaces of normal erythrocytes, as previously reported, but that it extends to erythroid precursor cells in the bone marrow of anemic malaria patients. Monoclonal mouse antibodies and human sera from patients with severe anemia, reacting with RSP2-tagged erythrocytes, induced cell destruction by phagocytosis and complement activation in vitro. Our observations reveal a new parasite mechanism implicated in the destruction of normal erythrocytes and probably dyserythropoiesis in malaria patients. These data suggest that the tagging of host cells with RSP2 may trigger anemia in falciparum malaria.

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.

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.

Placenta cryosections for study of the adhesion of Plasmodium falciparum-infected erythrocytes to chondroitin sulfate A in flow conditions.

The adhesion of Plasmodium falciparum-infected erythrocytes (IEs) to chondroitin-4-sulfate (CSA) via the PfEMP1-CSA parasite ligand domain is correlated with placental malaria in primigravidae. The recent identification of parasite genes encoding CSA adhesion molecules and the development of pan-reactive monoclonal antibodies against the Pf(CSA) ligand have opened up new avenues for the development of anti-IE sequestration therapies for the prevention of placental malaria. A model closely mimicking placental sequestration of IEs during pregnancy is needed for the preclinical and clinical evaluation of candidate molecules for the induction of antibodies that could protect pregnant women from placental malaria. We found that normal placenta cryosections were a specific and highly consistent support for the binding of IEs to CSA in flow conditions under physiological conditions. This model makes possible the quantitative and qualitative analysis of IE adhesion. We identified distinct CSA-binding phenotypes within the FCR3(CSA)-selected parasites in flow analyses, but not in static analyses. We also analyzed inhibitors of placental parasite binding such as soluble CSA and antibodies directed against the Pf(CSA) ligand. Our data demonstrate that placenta cryosections could be used to standardize assays between laboratories, potentially advancing the development of therapies against placental malaria.

Platelets reorient Plasmodium falciparum-infected erythrocyte cytoadhesion to activated endothelial cells.

Severe malaria is characterized by the sequestration of Plasmodium falciparum-infected erythrocytes (IEs). Because platelets can affect tumor necrosis factor (TNF)-activated endothelial cells (ECs), we investigated their role in the sequestration of IEs, using IEs that were selected because they can adhere to endothelial CD36 (IE(CD36)), a P. falciparum receptor that is expressed on platelets. The results of coincubation studies indicated that platelets can induce IE(CD36) binding to CD36-deficient brain microvascular ECs. This induced cytoadhesion resisted physiological shear stress, was increased by EC stimulation with TNF, and was abolished by anti-CD36 monoclonal antibody. Immunofluorescence and scanning electron microscopy results showed that platelets serve as a bridge between IEs and the surface of ECs and may therefore provide receptors for adhesion to microvascular beds that otherwise lack adhesion receptors. This novel mechanism of cytoadhesion may reorient the sequestration of different parasite phenotypes and play an important role in the pathogenesis of severe malaria.

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.

Immunization with recombinant duffy binding-like-gamma3 induces pan-reactive and adhesion-blocking antibodies against placental chondroitin sulfate A-binding Plasmodium falciparum parasites.

Maternal malaria is associated with the sequestration, in the placenta, of Plasmodium falciparum-infected erythrocytes onto chondroitin sulfate A (CSA), via the duffy binding-like (DBL)-gamma3 domain of the P. falciparum erythrocyte membrane protein 1 (PfEMP1(CSA)) (DBL-gamma3(CSA)). The production of antibodies against CSA-binding infected erythrocytes (IEs(CSA)) is correlated with resistance to maternal malaria in multiparous women. We produced recombinant DBL-gamma3(CSA) (rDBL-gamma3(CSA)) in insect cells, corresponding to 2 variant DBL-gamma3(CSA) subtypes that mediate binding to CSA in laboratory lines and placental isolates. Both recombinant cysteine-rich DBL-gamma3(CSA) domains blocked IEs(CSA) binding to CSA. Immunization of mice, with the rDBL-gamma3(CSA)-FCR3 and rDBL-gamma3(CSA)-3D7 domains, resulted in the generation of antibodies recognizing homologous and heterologous rDBL-gamma3(CSA), a finding indicating conserved epitopes inducing a pan-reactive immune response. Mouse monoclonal antibodies (MAbs) against both recombinant proteins were pan-reactive with various IEs(CSA). One MAb efficiently inhibited and reversed IE(CSA) cytoadhesion to endothelial cells in vitro. Thus, DBL-gamma3(CSA) is the target of inhibitory and pan-reactive antibodies. Saimiri sciureus monkeys immunized with FCR3-rDBL-gamma3(CSA) developed pan-reactive and inhibitory antibodies, a finding suggesting that the development of a vaccine to prevent maternal malaria is feasible.

Adhesion of normal and Plasmodium falciparum ring-infected erythrocytes to endothelial cells and the placenta involves the rhoptry-derived ring surface protein-2.

Recent findings have challenged the current view of Plasmodium falciparum (P falciparum) blood-stage biology by demonstrating the cytoadhesion of early ring-stage-infected erythrocytes (rIEs) to host endothelial cells and placental syncytiotrophoblasts. The adhesion of rIEs was observed only in parasites that bind to the placenta via chondroitin sulfate A (CSA). In this work, a panel of mouse monoclonal antibodies (mAbs) that specifically inhibit cytoadhesion of rIEs but not of mature IEs was generated The previously described ring surface protein 2 (RSP-2), a 42-kDa protein, was identified as the target of the ring-stage-specific mAbs. Time course surface fluorescence experiments revealed a short overlap (approximately 4 hours) of expression between RSP-2 and P falciparum erythrocyte membrane protein 1 (PfEMP1). Their consecutive expression enables IEs to adhere to endothelial cells during the entire blood-stage cycle. During this study, a new phenotype was detected in parasite cultures, the adhesion of normal erythrocytes (nEs) to endothelial cells. All adherent nEs were coated with RSP-2. Immunolocalization studies show that RSP-2 is a rhoptry-derived protein that is discharged onto the erythrocyte membrane during contact with merozoites. Our results identify RSP-2 as a key molecule in sequestration of young blood-stage forms and nEs to endothelial cells.