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

Antibodies to a Full-Length VAR2CSA Immunogen Are Broadly Strain-Transcendent but Do Not Cross-Inhibit Different Placental-Type Parasite Isolates

The high molecular weight, multidomain VAR2CSA protein mediating adhesion of Plasmodium falciparum-infected erythrocytes in the placenta is the leading candidate for a pregnancy malaria vaccine. However, it has been difficult so far to generate strong and consistent adhesion blocking antibody responses against most single-domain VAR2CSA immunogens. Recent advances in expression of the full-length recombinant protein showed it binds with much greater specificity and affinity to chondroitin sulphate A (CSA) than individual VAR2CSA domains. This raises the possibility that a specific CSA binding pocket(s) is formed in the full length antigen and could be an important target for vaccine development. In this study, we compared the immunogenicity of a full-length VAR2CSA recombinant protein containing all six Duffy binding-like (DBL) domains to that of a three-domain construct (DBL4-6) in mice and rabbits. Animals immunized with either immunogen acquired antibodies reacting with several VAR2CSA individual domains by ELISA, but antibody responses against the highly conserved DBL4 domain were weaker in animals immunized with full-length DBL1-6 recombinant protein compared to DBL4-6 recombinant protein. Both immunogens induced cross-reactive antibodies to several heterologous CSA-binding parasite lines expressing different VAR2CSA orthologues. However, antibodies that inhibited adhesion of parasites to CSA were only elicited in rabbits immunized with full-length immunogen and inhibition was restricted to the homologous CSA-binding parasite. These findings demonstrate that partial and full-length VAR2CSA immunogens induce cross-reactive antibodies, but inhibitory antibody responses to full-length immunogen were highly allele-specific and variable between animal species

Down-selection of the VAR2CSA DBL1-2 expressed in E. coli as a lead antigen for placental malaria vaccine development

International audienceOver 50 million women are exposed to the risk of malaria during pregnancy every year. Malaria during pregnancy is a leading global cause of maternal morbidity and adverse pregnancy outcomes. Adhesion of Plasmodium falciparum-infected erythrocytes to placental chondroitin-4-sulfate (CSA) has been linked to the severe disease outcome of placental malaria. Accumulated evidence strongly supports VAR2CSA as the leading placental malaria vaccine candidate. Recombinant proteins encompassing the VAR2CSA high affinity CSA binding site have been generated, and their activity as immunogens that elicit functional (inhibitory) and crossreactive antibodies against CSA-binding parasites assessed. The expression of His-tagged proteins was compared in four different expression systems and their capacity to bind specifically to CSA was analyzed. CHO cells and E. coli SHuffle cells were the two expression systems able to express some of the recombinant proteins in reasonable amounts. Larger analytical scale production of DBL1x-2× (3D7) and DBL3x-4ε (FCR3) best expressed in CHO and E. coli SHuffle cells were performed. Purified proteins were administered to rats either alone or adjuvanted with human approved adjuvants. Analysis of the functionality and cross-reactivity of the induced antibodies allowed us to down-select the DBL1x-2(3D7) expressed in E. coli SHuffle cells as the best antigen to be transitioned to further clinical development in order to protect future pregnant women living in malaria endemic areas against the severe clinical outcomes of placental malaria

VAR2CSA binding phenotype has ancient origin and arose before Plasmodium falciparum crossed to humans: implications in placental malaria vaccine design

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Heterozygous HbAC but not HbAS is associated with higher newborn birthweight among women with pregnancy-associated malaria

International audiencePregnancy-associated malaria (PAM) is associated with poor pregnancy outcomes. Hemoglobin S (HbS) and hemoglobin C (HbC) mutations are frequently encountered in malaria-endemic areas of Africa, where they protect children from severe and uncomplicated Plasmodium falciparum malaria. However, scant epidemiological data exist on the impact of these Hb variants on PAM. A prospective cohort of 635 Beninese pregnant women was recruited before 24 weeks of gestational age and followed until the end of pregnancy. HbAA, HbAC, and HbAS genotypes were determined and tested for association with pregnancy outcomes and PAM indicators using linear and logistic multivariate models. Newborns from HbAC mothers had higher birthweights than those from HbAA mothers among women infected at any time during pregnancy (mean difference 182.9 g, p = 0.08), or during the first half of pregnancy (654.3 g, p = 0.0006). No such birthweight differences were observed between newborns from HbAS and HbAA mothers. HbAC and HbAS were not associated with other pregnancy outcomes or PAM indicators. In conclusion, HbAC but not HbAS is associated with an improved birth outcome in pregnant women with documented PAM. Higher-birthweight newborns from HbAC mothers may have a survival advantage that contributes to the natural selection of HbC in malaria-endemic areas

Influence of the timing of malaria infection during pregnancy on birth weight and on maternal anemia in Benin

Although consequences of malaria in pregnancy are well known, the period of pregnancy in which infection has the highest impact is still unclear. In Benin, we followed up a cohort of 1,037 women through pregnancy until delivery. The objective was to evaluate the relationship between the timing of infection and birth weight, and maternal anemia at delivery. At the beginning of pregnancy, peripheral infections were associated with a decrease in mean birth weight (-98.5 g; P = 0.03) and an increase in the risk of anemia at delivery (adjusted odds ratio [aOR] = 1.6; P = 0.03). Infections in late pregnancy were related to a higher risk of maternal anemia at delivery (aOR = 1.7; P = 0.001). To fully protect the women during the whole pregnancy, already implemented measures (insecticide-treated nets and intermittent preventive treatment) should be reinforced. In the future, a vaccine against pregnancy-associated malaria parasites could protect the women in early pregnancy, which seems to be a high-risk period