Justin Doritchamou

The NTS-DBL2X Region of VAR2CSA Induces Cross-Reactive Antibodies That Inhibit Adhesion of Several Plasmodium falciparum Isolates to Chondroitin Sulfate A

BACKGROUND Binding to chondroitin sulfate A by VAR2CSA, a parasite protein expressed on infected erythrocytes, allows placental sequestration of Plasmodium falciparum-infected erythrocytes. This leads to severe consequences such as maternal anemia, stillbirths, and intrauterine growth retardation. The latter has been clearly associated to increased morbidity and mortality of the infants. Acquired anti-VAR2CSA antibodies have been associated with improved pregnancy outcomes, suggesting a vaccine could prevent the syndrome. However, identifying functionally important regions in the large VAR2CSA protein is difficult. METHODS Using genetic immunization, we raised polyclonal antisera against overlapping segments of VAR2CSA in mice and rabbits. The adhesion-inhibition capacities of induced antisera and of specific antibodies purified from plasma of malaria-exposed pregnant women were assessed on laboratory-adapted parasite lines and field isolates expressing VAR2CSA. Competition enzyme-linked immunosorbent assay (ELISA) was employed to analyze functional resemblance between antibodies induced in animals and those naturally acquired by immune multigravidae. RESULTS Antibodies targeting the N-terminal sequence (NTS) up to DBL2X (NTS-DBL2X) efficiently blocked parasite adhesion to chondroitin sulfate A in a manner similar to that of antibodies raised against the entire VAR2CSA extracellular domain. Interestingly, naturally acquired antibodies and those induced by vaccination against NTS-DBL2X target overlapping strain-transcendent anti-adhesion epitopes. CONCLUSIONS This study highlights an important step achieved toward development of a protective vaccine against placental malaria.

Comparison of Sulfadoxine‐Pyrimethamine, Unsupervised Artemether‐Lumefantrine, and Unsupervised Artesunate‐Amodiaquine Fixed‐Dose Formulation for Uncomplicated Plasmodium falciparum Malaria in Benin: A Randomized Effectiveness Noninferiority Trial

BACKGROUND We compared sulfadoxine-pyrimethamine (SP) with unsupervised artemether-lumefantrine (AL) and unsupervised amodiaquine-artesunate (ASAQ) fixed-dose formulation for the treatment of uncomplicated malaria in children in Benin. METHODS This open-label, noninferiority comparative trial included children aged 6-60 months. The follow-up period was 6 weeks, and the primary objective was a comparison of polymerase chain reaction (PCR)-adjusted effectiveness rates at day 28. RESULTS The study included 240 children (48 received SP, and 96 each received AL and ASAQ). The intention-to-treat analysis showed effectiveness rates on day 28 of 20.8%, 78.1%, and 70.5% for SP, AL, and ASAQ, respectively. After adjustment for PCR results, these rates were 27.1%, 83.3%, and 87.4%, respectively. The per-protocol analysis (217 patients) showed effectiveness rates on day 28 of 21.7%, 88.0%, and 76.1% for SP, AL, and ASAQ, respectively. After adjustment for PCR results, these rates were 28.3%, 94.0%, and 93.2%, respectively. SP was less effective than the other drugs in the PCR-adjusted analysis, whereas AL and ASAQ were equally effective. The rate of new infection was higher among children treated with ASAQ than among those treated with AL. CONCLUSIONS This was the first trial, to our knowledge, to compare unsupervised AL with unsupervised ASAQ fixed-dose formulation; both treatments provided high PCR-adjusted day 28 effectiveness rates. Efficacy rates for SP were surprisingly low. Clinical trials registration. NCT00460369.

High efficacy of anti DBL4ɛ-VAR2CSA antibodies in inhibition of CSA-binding Plasmodium falciparum-infected erythrocytes from pregnant women.

Malaria during pregnancy is a major cause of intra-uterine growth-retardation and infant death in sub-Saharan Africa. Ideally, this could be prevented by a vaccine delivered before the first pregnancy. Antibodies against domain DBL4ɛ from VAR2CSA has been shown to inhibit adhesion of laboratory isolates to the placental receptor chondroitin sulfate A. In this study, the binding inhibitory efficacy of IgG elicited by two different DBL4ɛ recombinant proteins was tested on a panel of fresh clinical isolates from pregnant women living in Benin and Tanzania. The most promising recombinant protein elicited antibodies with similar efficacy as pooled plasma from immune multi-gravid African women.

First-Trimester Plasmodium falciparum Infections Display a Typical “Placental” Phenotype

BACKGROUND Plasmodium falciparum-infected erythrocytes (IEs) adhere to host cell receptors, allowing parasites to sequester into deep vascular beds of various organs. This defining phenomenon of malaria pathogenesis is key to the severe clinical complications associated with cerebral and placental malaria. The principal ligand associated with the binding to chondroitin sulfate A (CSA) that allows placental sequestration of IEs is a P. falciparum erythrocyte membrane protein 1 (PfEMP1) family member encoded by the var2csa gene. METHODS Here, we investigated the transcription pattern of var genes by real-time polymerase chain reaction, the expression of VAR2CSA, protein by flow cytometry, and the CSA-binding ability of IEs collected at different stages of pregnancy using a static-based Petri dish assay. RESULTS Through comparison with the profiles of isolates from nonpregnant hosts, we report several lines of evidence showing that parasites infecting women during pregnancy preferentially express VAR2CSA protein, and that selection for the capacity to adhere to CSA via VAR2CSA expression occurs early in pregnancy. CONCLUSIONS Our data suggest that the placental tropism of P. falciparum is already established in the first trimester of pregnancy, with consequent implications for the development of the pathology associated with placental malaria.

Identification of Id1-DBL2X of VAR2CSA as a key domain inducing highly inhibitory and cross-reactive antibodies

PURPOSE OF THE RESEARCH VAR2CSA is considered as the main target of protective immunity against pregnancy-associated malaria. VAR2CSA high molecular weight complicates scaling up production of VAR2CSA recombinant protein for large-scale vaccination programmes. We previously demonstrated that antibodies induced by NTS-DBL1X-Id1-DBL2X efficiently block parasite binding to CSA in a similar manner to antibodies induced by the full-length extracellular part of VAR2CSA. In order to identifying the shortest fragment of VAR2CSA carrying major protective epitopes able to elicit inhibitory antibodies, we performed a refined antigenic mapping of NTS-DBL1X-Id1-DBL2X through a DNA vaccination technique. PRINCIPAL RESULTS Five single or double domains constructs encoding NTS-DBL1X, NTS-DBL1X-Id1, Id1, Id1-DBL2X and DBL2X were made and used to immunize mice. The NTS-DBL1X, NTS-DBL1X-Id1, and Id1-DBL2X fragments all raised high titer immune response, as measured by ELISA. The DBL2X fragment raised a weaker antibody titer, and the Id1 construct failed to elicit antibody. Sera from mice immunized with NTS-DBL1X or DBL2X constructs failed to block infected erythrocytes binding to CSA, whereas sera from mice immunized with NTS-DBL1X-Id1 showed partial inhibitory activity, and the Id1-DBL2X fragment elicited antisera that totally abrogated infected erythrocytes adhesion to CSA. IgG purified from Id1-DBL2X antisera showed a similar inhibitory profile than Id1-DBL2X antisera. Anti-FCR3 anti-Id1-DBL2X antibodies also efficiently block the adhesion of erythrocytes infected by the HB3 parasite line to CSA. Id1-DBL2X antisera recognized the surface of field isolates from pregnant women, and inhibited CSA-binding of all 8 isolates tested, although to a variable level. MAJOR CONCLUSIONS We raised high-titer antibodies against several parts of the protein, and identified Id1-DBL2X as the minimal VAR2CSA fragment inducing antibodies with CSA-binding inhibitory efficiency in the same range as the full-length extracellular part of VAR2CSA.

Protective Antibodies against Placental Malaria and Poor Outcomes during Pregnancy, Benin

Immunity requires a vaccine that inhibits binding of infected erythrocytes to chondroitin sulfate.

Imbalanced Distribution of GM Immunoglobulin Allotypes According to the Clinical Presentation of Plasmodium falciparum Malaria in Beninese Children

Selection pressure exerted by pathogens contributes to the persistence of polymorphisms in GM and KM allotypes, which are antigenic determinants of immunoglobulins. This study investigated the impact of GM and KM allotypes on the clinical response to Plasmodium falciparum infection among Beninese children, including 65 with severe malaria, 37 with uncomplicated malaria, and 53 with asymptomatic carriage. An inverse relationship was found between the GM 5,6,13,14; 1,17 phenotype and uncomplicated malaria. Genetic markers implicated in the composition and activity of immunoglobulins may be associated with the genetic control of both malaria infection and morbidity.

Functional Antibodies against VAR2CSA in Nonpregnant Populations from Colombia Exposed to Plasmodium falciparum and Plasmodium vivax

ABSTRACT In pregnancy, parity-dependent immunity is observed in response to placental infection with Plasmodium falciparum. Antibodies recognize the surface antigen, VAR2CSA, expressed on infected red blood cells and inhibit cytoadherence to the placental tissue. In most settings of malaria endemicity, antibodies against VAR2CSA are predominantly observed in multigravid women and infrequently in men, children, and nulligravid women. However, in Colombia, we detected antibodies against multiple constructs of VAR2CSA among men and children with acute P. falciparum and Plasmodium vivax infection. The majority of men and children (>60%) had high levels of IgGs against three recombinant domains of VAR2CSA: DBL5ε, DBL3X, and ID1-ID2. Surprisingly, these antibodies were observed only in pregnant women, men, and children exposed either to P. falciparum or to P. vivax. Moreover, the anti-VAR2CSA antibodies are of high avidity and efficiently inhibit adherence of infected red blood cells to chondroitin sulfate A in vitro, suggesting that they are specific and functional. These unexpected results suggest that there may be genotypic or phenotypic differences in the parasites of this region or in the host response to either P. falciparum or P. vivax infection outside pregnancy. These findings may hold significant clinical relevance to the pathophysiology and outcome of malaria infections in this region.

Differential adhesion-inhibitory patterns of antibodies raised against two major variants of the NTS-DBL2X region of VAR2CSA

BACKGROUND VAR2CSA is a large polymorphic Plasmodium falciparum protein expressed on infected erythrocytes (IE) that allows their binding in the placenta, thus precipitating placental malaria (PM). The N-terminal part of VAR2CSA that contains the binding site to placental chondroitin sulfate A (CSA) is currently recognized as the most attractive region for vaccine development. An ultimate challenge is to define epitopes in this region that induce a broad cross-reactive adhesion inhibitory antibody response. METHODS Based on phylogenetic data that identified a dimorphic sequence motif in the VAR2CSA DBL2X, we raised antibodies against the NTS-DBL2X constructs containing one sequence or the other (3D7 and FCR3) and tested their functional properties on P. falciparum isolates from pregnant women and on laboratory-adapted strains. RESULTS The CSA binding inhibitory capacity of the antibodies induced varied from one parasite isolate to another (range, 10%–100%), but the combined analysis of individual activity highlighted a broader functionality that increased the total number of isolates inhibited. Interestingly, the differential inhibitory effect of the antibodies observed on field isolates resulted in significant inhibition of all field isolates tested, suggesting that optimal inhibitory spectrum on field isolates from pregnant women might be achieved with antibodies targeting limited variants of the N-terminal VAR2CSA. CONCLUSIONS Our findings indicate that the NTS-DBL2X region of VAR2CSA can elicit strain-transcending anti-adhesion antibodies and suggest that the combination of the two major variants used here could represent the basis for an effective bivalent VAR2CSA-based vaccine.

Dynamics in the Cytoadherence Phenotypes of Plasmodium falciparum Infected Erythrocytes Isolated during Pregnancy

Pregnant women become susceptible to malaria infection despite their acquired immunity to this disease from childhood. The placental sequestration of Plasmodium falciparum infected erythrocytes (IE) is the major feature of malaria during pregnancy, due to ability of these parasites to bind chondroitin sulfate A (CSA) in the placenta through the VAR2CSA protein that parasites express on the surface of IE. We collected parasites at different times of pregnancy and investigated the adhesion pattern of freshly collected isolates on the three well described host receptors (CSPG, CD36 and ICAM-1). Var genes transcription profile and VAR2CSA surface-expression were assessed in these isolates. Although adhesion of IE to CD36 and ICAM-1 was observed in some isolates, CSA-adhesion was the predominant binding feature in all isolates analyzed. Co-existence in the peripheral blood of several adhesion phenotypes in early pregnancy isolates was observed, a diversity that gradually tightens with gestational age in favour of the CSA-adhesion phenotype. Infections occurring in primigravidae were often by parasites that adhered more to CSA than those from multigravidae. Data from this study further emphasize the specificity of CSA adhesion and VAR2CSA expression by parasites responsible for pregnancy malaria, while drawing attention to the phenotypic complexity of infections occurring early in pregnancy as well as in multigravidae.