Identification of Plasmodium falciparum var1CSA and var2CSA domains that bind IgM natural antibodies

Malaria in pregnancy is responsible for maternal anaemia, low-birth-weight babies and infant deaths. Plasmodium falciparum infected erythrocytes are thought to cause placental pathology by adhering to host receptors such as chondroitin sulphate A (CSA). CSA binding infected erythrocytes also bind IgM natural antibodies from normal human serum, a process that may facilitate placental adhesion or promote immune evasion. The parasite ligands that mediate placental adhesion are thought to be members of the variant erythrocyte surface antigen family P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var genes. Two var gene sub-families, var1CSA and var2CSA, have been identified as parasite CSA binding ligands and are leading candidates for a vaccine to prevent pregnancy-associated malaria. We investigated whether these two var gene subfamilies implicated in CSA binding are also the molecules responsible for IgM natural antibody binding. By heterologous expression of domains in COS-7 cells, we found that both var1CSA and var2CSA PfEMP1 variants bound IgM, and in both cases the binding region was a DBL epsilon domain occurring proximal to the membrane. None of the domains from a control non-IgM-binding parasite (R29) bound IgM when expressed in COS-7 cells. These results show that PfEMP1 is a parasite ligand for non-immune IgM and are the first demonstration of a specific adhesive function for PfEMP1 epsilon type domains

Impact des polluants et du changement climatique sur les capacités de reproduction et le développement embryo larvaire de l’huître creuse Crassostrea gigas dans le Bassin d’Arcachon

The Pacific oyster represents an important part of the aquaculture production, with 555 913 tons produced in 2013. France is the fourth world producer of oysters and Crassostrea gigas is the principal cultivated species in the Arcachon bay. However, in recent years, problems of recruitment, capture and mortality of juvenile or adult oysters occurred. The increased frequency of these events can be indicative of changes in the global quality of the Arcachon bay. In this context of crisis, this work has been focused on the impact of two pollutants, copper and S-metolachlor, mostly found in the waters of the Arcachon bay, on D-larvae embryo-larval development of the Pacific oyster (24h post -fecundation). In a context of global climate change, a multifactorial approach was adopted to study the combined effects of pollutants and increase temperature or salinity changes that could affect the development and survival of embryos and larvae during reproduction season. First, the embryo-toxic effects of copper or S-metolachlor coupled to salinity or different temperatures were studied with oysters from hatchery. For this, the embryo-larval test was used, and its application limits specified. Secondly, the effects of pollutants combined or not to environmental temperature and salinity were analyzed on embryos from wild or cultivated oysters harvested directly on different sites of the Arcachon bay. The percentage of abnormalities and the differential expression levels of target genes were determined in larvae while the Cu and S-metolachlor bioaccumulation and the percentage of gonadal occupation were measured in genitors. These results were then compared to determine the ability of oyster from hatchery to represent an interesting alternative model to indigenous oysters. With the objective to study the impacts of climate change, somewhat more extreme conditions were tested, namely temperatures of 26 ° C, lower salinity of 24 u.s.i and higher concentrations of pollutants compared to current conditions in the lagoon. A software able to analyze swimming behavior of D-larvae was also developed. Our results indicate higher bioaccumulation of Cu and S-metolachlor in cultivated oysters compared to wild oysters. Furthermore this study clearly indicates that larvae from indigenous oysters (wild and cultivated) are sensitive to environmental concentrations of copper and S-metolachlor. However, it has been demonstrated that genes involved in various defense mechanisms are overexpressed with higher larval defense capacity of oysters under the influence of the tributaries but also wild oysters in comparison to cultivated oysters. In the presence of environmental concentrations of Cu or S-metolachlor, an increased erratic circular trajectory was found. In addition, larvae, although able to grow normally in a temperature range of 22 ° C to 26 ° C, are sensitive to the action of the combined high / low temperatures and pollutants. Similarly, they are sensitive to the combined effect of low salinity and exposure to pollutants. Oyster’s hatchery have proved to be a good alternative to the use of indigenous oysters. Finally, given the predictions of climate change, our results indicate that one can expect an increase in larval abnormalities and thus a decrease in the recruitment of spat in future years in the Arcachon bay.L'huître creuse du Pacifique constitue une part importante de la production aquacole mondiale, avec 555 913 tonnes produites en 2013. La France est le quatrième producteur mondial d'huîtres et Crassostrea gigas est la principale espèce cultivée dans le bassin d'Arcachon. Cependant, ces dernières années, des problèmes de recrutement et de captage des naissains de certaines cohortes d’huîtres sont survenus. L'augmentation de la fréquence de ces événements peut être révélatrice de changements dans la qualité du milieu. Dans ce contexte de crise, ces travaux se sont intéressés à l’impact de deux polluants, le cuivre et le S-métolachlore, majoritairement retrouvés dans les eaux du Bassin d’Arcachon sur le développement embryo-larvaire des larves D de l’huître creuse (24h post -fécondation). Dans un contexte de changement climatique, une approche multifactorielle a été adoptée afin d’étudier les effets combinés des polluants et de l’accroissement des températures ou des changements de salinité susceptibles d’altérer le développement et la survie des embryons et des larves en période estivale. Dans un premier temps, les effets embryo-toxiques d’une pollution par le cuivre ou le S-métolachlore couplés ou non à la salinité ou à différentes températures ont été étudiés sur des huîtres en provenance d’une écloserie. Pour cela le test embryo-larvaire a été utilisé, et ses limites d’application précisées. Dans un deuxième temps, les effets des polluants couplés ou non à des températures et salinités environnementales ont été analysés sur les embryons provenant d’huîtres sauvages ou cultivées prélevées directement dans le milieu en différents sites du Bassin d’Arcachon. Le pourcentage de malformations ainsi que l’expression différentielle de gènes cibles ont été déterminés chez les larves tandis que la bioaccumulation du cuivre et du S-métolachlore et le pourcentage d’occupation gonadique ont été analysés chez les géniteurs. Ces résultats ont ensuite été comparés afin de déterminer la capacité des huîtres d’écloserie à représenter un modèle alternatif aux huîtres autochtones. Dans l’objectif d’étudier les impacts liés au changement climatique, des conditions un peu plus extrêmes ont été testées, à savoir des températures supérieures de 26 °C, des salinités inférieures de 24 u.s.i et des concentrations en polluants supérieures aux conditions actuelles du bassin. La mise au point d’un logiciel d’analyse du comportement natatoire des larves D a également été réalisée. Nos résultats indiquent une bioaccumulation plus grande du Cu et S-métolachlore dans les huîtres cultivées par rapport aux huîtres sauvages. Par ailleurs cette étude indique clairement que les larves issues des huîtres autochtones (sauvages et cultivées) sont sensibles à des concentrations environnementales en cuivre et en S-métolachlore. Cependant, il a été montré que les gènes impliqués dans divers mécanismes de défense sont surexprimés, avec une plus grande capacité de défense des larves issues des huîtres sous l’influence des tributaires mais également des huîtres sauvages par rapport aux cultivées. En présence de concentrations environnementales de Cu comme de S-métolachlore, une augmentation des trajectoires erratiques circulaires a été constatée. De plus, les larves, bien que capables de se développer normalement dans une gamme de températures allant de 22 °C à 26 °C, sont sensibles à l’action combinées des hautes/basses températures et des polluants. De la même manière, elles sont sensibles aux effets combinés de la dessalure et de l’exposition aux polluants. Les huîtres d’écloserie se sont révélées être une bonne alternative à l’utilisation des huîtres autochtones. Finalement, au vu des prédictions concernant l’évolution du climat, nos résultats indiquent qu’il faut s’attendre à un accroissement des malformations larvaires et donc à une diminution du recrutement des naissains dans les années futures dans le Bassin d’Arcachon

Do Temporal and Spatial Parameters or Lifestyle of the Pacific Oyster Crasssostrea gigas Affect Pollutant Bioaccumulation, Offspring Development, and Tolerance to Pollutants?

International audienceThis study evaluated the vulnerability of early life stages of native oysters (Crassostrea gigas) from the Arcachon Bay (SW, France) to pollutants at risk in the lagoon in particular copper and S-metolachlor. Developmental abnormalities in wild and cultivated oyster D-larvae were investigated during 2 breeding-seasons (2013 and 2014) at different sampling sites and dates. In addition, copper, and metolachlor concentrations were determined both in seawater and in mature oysters. Bioaccumulation of Cu was observed at higher levels in wild than in farmed specimens. Metolachlor was accumulated at much lower levels. After 24 h exposure, significant increases of the percentage of abnormal D-larvae were observed when exposed at 1 μg L−1 of copper or 10 ng L−1 of metolachlor in comparison with the controls whatever the date, the site, and rearing conditions of the genitors. The current study demonstrates that environmental concentrations of copper and metolachlor can induce a significant increase of developmental abnormalities in farmed and wild populations of oysters. However, no significant differences of sensitivity were observed according to temporal, spatial parameters, and lifestyle of genitors. In addition, oyster larvae obtained from the hatchery displayed the same sensitivity to pollutants as larvae from the field, demonstrating their suitability for toxicity assays and water quality monitoring

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

Var2CSA Minimal CSA Binding Region Is Located within the N-Terminal Region

Var2CSA, a key molecule linked with pregnancy-associated malaria (PAM), causes sequestration of Plasmodium falciparum infected erythrocytes (PEs) in the placenta by adhesion to chondroitin sulfate A (CSA). Var2CSA possesses a 300 kDa extracellular region composed of six Duffy-binding like (DBL) domains and a cysteine-rich interdomain region (CIDRpam) module. Although initial studies implicated several individual var2CSA DBL domains as important for adhesion of PEs to CSA, new studies revealed that these individual domains lack both the affinity and specificity displayed by the full-length extracellular region. Indeed, recent evidence suggests the presence of a single CSA-binding site formed by a higher-order domain organization rather than several independent binding sites located on the different domains. Here, we search for the minimal binding region within var2CSA that maintains high affinity and specificity for CSA binding, a characteristic feature of the full-length extracellular region. Accordingly, truncated recombinant var2CSA proteins comprising different domain combinations were expressed and their binding characteristics assessed against different sulfated glycosaminoglycans (GAGs). Our results indicate that the smallest region within var2CSA with similar binding properties to those of the full-length var2CSA is DBL1X-3X. We also demonstrate that inhibitory antibodies raised in rabbit against the full-length DBL1X-6ε target principally DBL3X and, to a lesser extent, DBL5ε. Taken together, our results indicate that efforts should focus on the DBL1X-3X region for developing vaccine and therapeutic strategies aimed at combating PAM

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

Human pregnancy-associated malaria-specific B cells target polymorphic, conformational epitopes in VAR2CSA

Pregnancy-associated malaria (PAM) is caused by Plasmodium falciparum-infected erythrocytes (IEs) that bind to chondroitin sulphate A (CSA) in the placenta by PAM-associated clonally variant surface antigens (VSA). Pregnancy-specific VSA (VSAPAM), which include the PfEMP1 variant VAR2CSA, are targets of IgG-mediated protective immunity to PAM. Here, we report an investigation of the specificity of naturally acquired immunity to PAM, using eight human monoclonal IgG1 antibodies that react exclusively with intact CSA-adhering IEs expressing VSAPAM. Four reacted in Western blotting with high-molecular-weight (> 200 kDa) proteins, while seven reacted with either the DBL3-X or the DBL5-ε domains of VAR2CSA expressed either as Baculovirus constructs or on the surface of transfected Jurkat cells. We used a panel of recombinant antigens representing DBL3-X domains from P. falciparum field isolates to evaluate B-cell epitope diversity among parasite isolates, and identified the binding site of one monoclonal antibody using a chimeric DBL3-X construct. Our findings show that there is a high-frequency memory response to VSAPAM, indicating that VAR2CSA is a primary target of naturally acquired PAM-specific protective immunity, and demonstrate the value of human monoclonal antibodies and conformationally intact recombinant antigens in VSA characterization

Clinical development of placental malaria vaccines and immunoassays harmonization:a workshop report

International audiencePlacental malaria caused by Plasmodium falciparum infection constitutes a major health problem manifesting as severe disease and anaemia in the mother, impaired fetal development, low birth weight or spontaneous abortion. Prevention of placental malaria currently relies on two key strategies that are losing efficacy due to spread of resistance: long-lasting insecticide-treated nets and intermittent preventive treatment during pregnancy. A placental malaria vaccine would be an attractive, cost-effective complement to the existing control tools. Two placental malaria vaccine candidates are currently in Phase Ia/b clinical trials. During two workshops hosted by the European Vaccine Initiative, one in Paris in April 2014 and the other in Brussels in November 2014, the main actors in placental malaria vaccine research discussed the harmonization of clinical development plans and of the immunoassays with a goal to define standards that will allow comparative assessment of different placental malaria vaccine candidates. The recommendations of these workshops should guide researchers and clinicians in the further development of placental malaria vaccines

Differential Recognition of P. falciparum VAR2CSA Domains by Naturally Acquired Antibodies in Pregnant Women from a Malaria Endemic Area

Plasmodium falciparum infected red blood cells (iRBC) express variant surface antigens (VSA) of which VAR2CSA is involved in placental sequestration and causes pregnancy-associated malaria (PAM). Primigravidae are most susceptible to PAM whereas antibodies associated with protection are often present at higher levels in multigravid women. However, HIV co-infection with malaria has been shown to alter this parity-dependent acquisition of immunity, with more severe symptoms as well as more malaria episodes in HIV positive women versus HIV negative women of a similar parity.Using VAR2CSA DBL-domains expressed on the surface of CHO-745 cells we quantified levels of DBL-domain specific IgG in sera from pregnant Malawian women by flow cytometry. Dissociations constants of DBL5epsilon specific antibodies were determined using a surface plasmon resonance technique, as an indication of antibody affinities.VAR2CSA DBL5epsilon was recognized in a gender and parity-dependent manner with anti-DBL5epsilon IgG correlating significantly with IgG levels to VSA-PAM on the iRBC surface. HIV positive women had lower levels of anti-DBL5epsilon IgG than HIV negative women of similar parity. In primigravidae, antibodies in HIV positive women also showed significantly lower affinity to VAR2CSA DBL5epsilon.Pregnant women from a malaria-endemic area had increased levels of anti-DBL5epsilon IgG by parity, indicating this domain of VAR2CSA to be a promising vaccine candidate against PAM. However, it is important to consider co-infection with HIV, as this seems to change the properties of antibody response against malaria. Understanding the characteristics of antibody response against VAR2CSA is undoubtedly imperative in order to design a functional and efficient vaccine against PAM

HIV infection and placental malaria reduce maternal transfer of multiple antimalarial antibodies in Mozambican women

Objectives: Maternal Plasmodium falciparum-specific antibodies may contribute to protect infants against severe malaria. Our main objective was to evaluate the impact of maternal HIV infection and placental malaria on the cord blood levels and efficiency of placental transfer of IgG and IgG subclasses.Methods: In a cohort of 341 delivering HIV-negative and HIV-positive mothers from southern Mozambique, we measured total IgG and IgG subclasses in maternal and cord blood pairs by quantitative suspension array technology against eight P. falciparum antigens: Duffy-binding like domains 3-4 of VAR2CSA from the erythrocyte membrane protein 1, erythrocyte-binding antigen 140, exported protein 1 (EXP1), merozoite surface proteins 1, 2 and 5, and reticulocyte-binding-homologue-4.2 (Rh4.2). We performed univariable and multivariable regression models to assess the association of maternal HIV infection, placental malaria, maternal variables and pregnancy outcomes on cord antibody levels and antibody transplacental transfer.Results: Maternal antibody levels were the main determinants of cord antibody levels. HIV infection and placental malaria reduced the transfer and cord levels of IgG and IgG1, and this was antigen-dependent. Low birth weight was associated with an increase of IgG2 in cord against EXP1 and Rh4.2.Conclusions: We found lower maternally transferred antibodies in HIV-exposed infants and those born from mothers with placental malaria, which may underlie increased susceptibility to malaria in these children