Jürg Gysin

Opsonization as an effector mechanism in human protection against asexual blood stages of Plasmodium falciparum: Functional role of IgG subclasses

A phagocytic assay performed with human peripheral mononuclear cells and malaria-infected erythrocytes enabled the study of opsonizing antibodies in human serum from donors presenting different levels of protective immunity. Opsonizing activity was found in sera from individuals who could be considered immune, i.e. asymptomatic parasite carriers and subjects residing in endemic regions who presented neither symptoms nor parasites. This contrasted with subjects showing an absence of symptoms or who had experienced only a single malarial infection. All sera contained high levels of antimalarial antibodies, as shown by immunofluorescence assay (IFA). IgG of different subclasses were immunopurified from these sera. All subclasses were shown to bind to the surface of infected erythrocytes by FACS analysis, but only IgG1 and IgG3 were able to mediate opsonization. IgG2 and IgG4 did not opsonize and inhibited the opsonizing activity of IgG1 and IgG3 in competition experiments. These results suggest the existence of a correlation between immune protection, the ability of serum to mediate opsonization of infected erythrocytes and the predominance, in this serum, of IgG1 and IgG3 over IgG2 and IgG4 directed against the surface of the infected erythrocytes.

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.

Chondroitin-4-sulphate (proteoglycan) a receptor for Plasmodium falciparum-infected erythrocyte adherence on brain microvascular endothelial cells

Adherence of Plasmodium falciparum parasitized erythrocytes to the microvascular endothelium is mediated by different receptors expressed by endothelial cells. The study of the adherence of P. falciparum-infected erythrocytes to Saimiri monkey brain microvascular endothelial cells revealed the presence of an additional receptor, which was identified and further characterized. This receptor was also found on the surface of primary human lung endothelial cells (HLEC). We developed two mAbs to this receptor which very efficiently blocked the adherence of parasite strains to Saimiri brain endothelial cells (SBEC). The ability of these mAb to bind to SBEC was partially blocked by chondroitin-4-sulphate (CSA). Competitive inhibition assays on adherence of parasitized red blood cells (PRBC) showed that CSA, but not hyaluronic acid, chondroitin-6-sulphate, dermatan sulphate, keratane sulphate, heparan sulphate or chondroitin-4S-disaccharide, was able to almost completely inhibit PRBC adherence. The same effect was obtained with chondroitinase ABC and AC, but not B, hyaluronidase or heparinase. These results strongly suggest that a member of the chondroitin-glycosaminoglycan family, CSA, represents an additional receptor used by P. falciparum PRBC to cytoadhere to microvascular endothelial cells.

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.

Protective antibodies against erythrocytic stages of Plasmodium falciparum in experimental infection of the squirrel monkey, Saimiri sciureus

Serum and ascitic fluid from squirrel monkeys (Saimiri sciureus) inoculated with erythrocytic stages of Plasmodium falciparum were collected at different periods of the infection. Protection against P. falciparum was achieved by passive transfer of the sera or fluid recovered from animals after spontaneous or drug‐induced cure. Purified immunoglobulins from the ascitic fluid also conferred protection. In contrast, protective antibodies directed against erythrocytic stages of P. falciparum could never be demonstrated during the acute phase of infection in spite of the high titres of malarial antibodies detected by immunofluorescence. The comparative immunochemical analysis of antigens recognized by protective and non‐protective antibodies revealed quantitative differences which may be of use for the identification of antigens inducing protection.

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.

Isolation and characterization of brain microvascular endothelial cells from Saimiri monkeys. An in vitro model for sequestration of Plasmodium falciparum-infected erythrocytes.

The adhesion of parasitized red blood cells (PRBC) to the endothelium (sequestration) may contribute to the pathogenic events in severe human malaria caused by P. falciparum. However, the factors involved in the pathophysiology, especially cerebral malaria are poorly understood. Previously, we have shown that the squirrel monkey Saimiri sciureus is a potential model for human cerebral malaria. In this paper we describe five stable clones of endothelial cell lines isolated immediately postmortem from different regions of the brain of Saimiri monkeys. The endothelial cell characteristics of these clones were confirmed by analyzing their ultrastructural aspects by transmission electron microscopy and by immunodetection of various endothelial cell markers. The Saimiri brain endothelial cell clones (SBEC) varied in their expression of different surface molecules. For example, various combinations of receptors involved in P. falciparum PRBC adherence such as CD36, ICAM-1 and E-selectin, were expressed at baseline values and could be up-regulated by human srTNF-alpha and human srIFN-gamma. One of the SBEC clones showed a strong cytoadherence for various laboratory strains of P. falciparum despite the absence of surface expression of any of the known endothelial receptors implicated in PRBC adherence. This finding suggests the existence of a new and uncharacterized PRBC binding receptor. The use of target organ specific endothelial cell lines expressing a number of different potential P. falciparum PRBC cytoadherence receptors, will be a useful in vitro system for the evaluation of strategies for the development of vaccine and antimalarial drugs to prevent human cerebral malaria.

Molecular mechanisms of Plasmodium falciparum placental adhesion

In natural Plasmodium falciparum infections, parasitized erythrocytes (PEs) circulate in the peripheral blood for a period corresponding roughly to the first part of the erythrocytic life cycle (ring stage). Later, in blood‐stage development, parasite‐encoded adhesion molecules are inserted into the erythrocyte membrane, preventing the circulation of the PEs. The principal molecule mediating PE adhesion is P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the polymorphic var gene family. The population of parasites is subject to clonal antigenic variation through changes in var expression, and a single PfEMP1 variant is expressed at the PE surface in a mutually exclusive manner. In addition to its role in immune evasion, switches in PfEMP1 expression may be associated with fundamental changes in parasite tissue tropism in malaria patients. A switch from CD36 binding to chondroitin sulphate A (CSA) binding may lead to extensive sequestration of PEs in placenta syncytiotrophoblasts. This is probably a key event in malaria pathogenesis during pregnancy. The CSA‐binding phenotype of mature PEs is linked to another distinct adhesive phenotype: the recently described CSA‐independent cytoadhesion of ring‐stage PEs. Thus, a subpopulation of PEs that sequentially displays these two different phenotypes may bind to an individual endothelial cell or syncytiotrophoblast throughout the asexual blood‐stage cycle. This suggests that non‐circulating (cryptic) parasite subpopulations are present in malaria patients.

Immunogenicity of Duffy Binding-Like Domains That Bind Chondroitin Sulfate A and Protection against Pregnancy-Associated Malaria

ABSTRACT 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.

Immunization with Recombinant Duffy Binding-Like-γ3 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.