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

<p>Abstract</p> <p>Background</p> <p>Pregnancy-associated malaria (PAM) is a serious consequence of <it>Plasmodium falciparum</it>-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.</p> <p>Methods</p> <p>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 <it>Escherichia coli </it>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-ε domains.</p> <p>Results</p> <p>Using the HEK293 expression system, DBL1-X, DBL4-ε and DBL6-ε were produced at relatively high levels in the culture supernatant, while DBL3-X and DBL5-ε were produced at much lower levels. DBL2-X and DBL3-X domains were obtained after refolding of the inclusion bodies produced in <it>E. coli</it>. Importantly, mice antisera raised against the recombinant DBL6-ε domain, specifically reacted against the surface of CSA-binding parasites and revealed adhesion blocking activity.</p> <p>Conclusion</p> <p>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.</p

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 FCR3Δvar2csa 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, FCR3Δvar2csa 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 FCR3Δvar2csa mutant parasites and highlight the key role of var2CSA in pregnancy malaria pathogenesis and for vaccine development

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

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-γ, 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-γ 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-γ 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-γ. 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

Etude du phénotype CSA dans le cadre du développement d'un vaccin contre le paludisme placentaire

Le but de ce travail a été d'identifier et de caractériser un ou plusieurs antigènes pouvant entrer dans le développement d'un vaccin contre le paludisme de la femme enceinte. Cet accès grave du paludisme est dû à la séquestration d'hématies parasitées (Hp) par Plasmodium falciparum de phénotype adhérant à la chondroitine sulfate A (CSA) dans le compartiment maternel du placenta. Nous avons développé des anticorps monoclonaux (acm) anti-var1CSA et anti-var2CSA spécifiquement dirigés contre les antigènes des Hp adhérant sur CSA. Ces acm sont capables d'inhiber la cytoadhérence des Hp de phénotype CSA, comme nous avons pu le démontrer grâce à un nouveau modèle de cytoadhérence des Hp sous flux sur des cryocoupes de placentas humains. Parmi les deux gènes varCSA connus, seule la transcription du gène var2CSA est augmentée dans les parasites placentaires. Afin d'analyser ces nouvelles données, nous avons produit des molécules recombinantes correspondant aux protéines parasitaires codées par les gènes varCSA (PfEMP1-var1CSA et -var2CSA) et prouvé leur fonctionnalité. En parallèle, grâce à nos acm inhibiteurs, nous avons identifié quatre domaines DBL du gène var2CSA qui pourraient être à l'origine de l'induction d'anticorps inhibiteurs de la cytoadhérence de ce phénotypeWe aimed to identify and to characterize one or several antigens of potential use for the development of a vaccine against pregnancy-associated malaria. This severe form of malaria results from the sequestration of Plasmodium falciparum-infected erythrocytes (IEs) adhering to chondroitin sulfate (CSA) in the maternal part of the placenta. We have developed monoclonal antibodies (mabs) against var1CSA and var2CSA specifically targeting the antigens of IEs with CSA-binding phenotypes, as demonstrated in a new model of IE cytoadhesion to cryosections of human placenta in flow conditions. The var2CSA gene is the only one of the two known var genes to display an increase in transcription in placental parasites. We have generated recombinant molecules corresponding to the parasite proteins encoded by the varCSA genes (PfEMP1-var1CSA and PfEMP1var2CSA) for further analyses and to demonstrate their functionality. In parallel, using our inhibitory mabs, we have identified four DBL domains of the var2CSA gene that may elicit antibodies inhibiting the cytoadhesion of IEs with this phenotypeAIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Paludisme grave (protéines de surface de l'hématie parasitée par P. Falciparum impliquées dans la séquestration parasitaire)

Les formes graves du paludisme sont associées à la séquestration parasitaire. Les protéines de surface des hématies parasitées (HP) sont à la fois les ligands de cette séquestration et les cibles de la réponse immunitaire. Elles représentent donc des candidats potentiels pour le développement d'une stratégie antipaludique. La cytoadhérence placentaire des HP matures est régie par l'interaction du DBLy3 de PfEMP1 avec la CSA alors que RSP2 est le ligand des formes jeunes. La production d'anticorps conformationnels contre les antigènes mineurs représente un obstacle majeur. Pour outrepasser cela, nous avons développé la technique d'immunisation des souris précédemment rendues tolérantes. Ainsi, nous avons produit des anticorps monoclonaux qui reconnaissent des conformations natives du DBLy3 (CSA) et RSP2. Après l'expression de DBLy3 (CSA) fonctionnelle dans le système d'expression des cellules d'insectes, nous avons produit des anticorps anti_DBLy3 (CSA) inhibant la cytoadhérence chez le singe et la souris. Ces anticorps nous ont permis de montrer que le DBL contient des épitopes conservés malgré sa diversité des séquences primaires. Les anticorps anti-RSP2 inhibent la cytoadhérence des formes jeunes des HP (CSA) et l'invasion des mérozoïtes. Ces anticorps nous ont permis de montrer que l'expression de RSP2 commence au stade trophozoïte, et qu'elle est accumulée dans les rhoptries avant d'être transportée à la surface du mérozoïte. RSP2 est transférée à la surface de l'hématie lorsque le mérozoïte s'y attache, sans que l'invasion ne soit nécessaire. Réunis, ces résultats promeuvent l'élaboration d'une stratégie thérapeutique anti- séquestration contre le paludisme gestationnel, et plus généralement contre la cytoadhérence des HP (CSA).PARIS12-CRETEIL BU Multidisc. (940282102) / SudocSudocFranceF

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 DBL3γ 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 DBLγ domains. Here, we demonstrate that antibodies raised against DBL3γ 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-rDBL3γ 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

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

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