Benoît Gamain

Publications (7)37.97 Total impact

• Article: Structural and immunological correlations between the variable blocks of the VAR2CSA domain DBL6ε from two Plasmodium falciparum parasite lines.

  Stéphane Gangnard, Cyril Badaut, Stéphanie Ramboarina, Bruno Baron, Tarik Ramdani, Benoît Gamain, Philippe Deloron, Anita Lewit-Bentley, Graham A Bentley
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  ABSTRACT: Plasmodium falciparum erythrocyte membrane protein (PfEMP1), a family of adhesins of the falciparum species of the malaria parasite, is exposed on the surface of the infected erythrocyte. In general, only one PfEMP1 variant is expressed at a time but switching between variants occurs, changing both host-cell receptor specificity and serotype. The PfEMP1 variant VAR2CSA causes sequestration of infected erythrocytes in the intervillous spaces of the placenta via the glycosaminoglycan chondroitin sulphate A. This leads to pregnancy-associated malaria, which has severe consequences for the foetus and mother. The extra-cellular region of VAR2CSA comprises six DBL domains and a single CIDR domain. The C-terminal domain DBL6ε, the most polymorphic domain of VAR2CSA, has seven regions of high variability termed variable blocks (VB). Here we have determined the crystal structure of DBL6ε from the FCR3 parasite line and have compared it with the previously determined structure of that from the 3D7 line. We found significant differences particularly in the N-terminal region, which contains the first VB (VB1). Although DBL6ε is the most variable VAR2CSA domain, DBL6ε-FCR3 and DBL6ε-3D7 react with IgG purified from immune sera of pregnant women. Furthermore, IgG purified on one domain cross-reacts with the other, confirming the presence of cross-reactive epitopes. We also examined reactivity of immune sera to the four least variable VB (VB1, VB2, VB4, VB5) using peptides with the consensus sequence closest, in turn, to the FCR3 or 3D7 domain. These results provide new molecular insights into immune escape by parasites expressing the VAR2CSA variant.
  Journal of Molecular Biology 02/2013; · 4.00 Impact Factor
• Article: Expressing Full-Length Functional PfEMP1 Proteins in the HEK293 Expression System.

  Anand Srivastava, Yves Durocher, Benoît Gamain
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  ABSTRACT: Due to the A/T-richness of the genome of Plasmodium falciparum, expressing P. falciparum proteins in heterologous expression systems is challenging. In addition, many P. falciparum proteins have high cysteine content and high molecular weight, which further complicates expression of these proteins in heterologous systems. The high molecular weight Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) adhesins expressed on the surface of the infected erythrocytes are among the most difficult proteins to express. Cost reduction in synthetic gene synthesis, as well as improved eukaryotic expression systems, now makes it possible to express such proteins. In this chapter, we describe the construction, production, purification, and functional assessment of the full-length extracellular region of the var2CSA PfEMP1 protein involved in pregnancy-associated malaria (PAM), using a human embryonic kidney (HEK) expression system.
  Methods in molecular biology (Clifton, N.J.) 01/2013; 923:307-19.
• Source

  Available from: Mirja Hommel

  Article: Antibodies to a full-length VAR2CSA immunogen are broadly strain-transcendent but do not cross-inhibit different placental-type parasite isolates.

  Marion Avril, Marianne J Hathaway, Anand Srivastava, Sébastien Dechavanne, Mirja Hommel, James G Beeson, Joseph D Smith, Benoît Gamain
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  ABSTRACT: 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.
  PLoS ONE 01/2011; 6(2):e16622. · 4.09 Impact Factor
• Source

  Available from: Graham Arthur Bentley

  Article: Var2CSA minimal CSA binding region is located within the N-terminal region.

  Anand Srivastava, Stéphane Gangnard, Sébastien Dechavanne, Farroudja Amirat, Anita Lewit Bentley, Graham A Bentley, Benoît Gamain
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  ABSTRACT: 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.
  PLoS ONE 01/2011; 6(5):e20270. · 4.09 Impact Factor
• Article: Full-length extracellular region of the var2CSA variant of PfEMP1 is required for specific, high-affinity binding to CSA.

  Anand Srivastava, Stéphane Gangnard, Adam Round, Sébastien Dechavanne, Alexandre Juillerat, Bertrand Raynal, Grazyna Faure, Bruno Baron, Stéphanie Ramboarina, Saurabh Kumar Singh, Hassan Belrhali, Patrick England, Anita Lewit-Bentley, Artur Scherf, Graham A Bentley, Benoît Gamain
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  ABSTRACT: Pregnancy-associated malaria (PAM) is a serious consequence of sequestration of Plasmodium falciparum-parasitized erythrocytes (PE) in the placenta through adhesion to chondroitin sulfate A (CSA) present on placental proteoglycans. Recent work implicates var2CSA, a member of the PfEMP1 family, as the mediator of placental sequestration and as a key target for PAM vaccine development. Var2CSA is a 350 kDa transmembrane protein, whose extracellular region includes six Duffy-binding-like (DBL) domains. Due to its size and high cysteine content, the full-length var2CSA extracellular region has not hitherto been expressed in heterologous systems, thus limiting investigations to individual recombinant domains. Here we report for the first time the expression of the full-length var2CSA extracellular region (domains DBL1X to DBL6epsilon) from the 3D7 parasite strain using the human embryonic kidney 293 cell line. We show that the recombinant extracellular var2CSA region is correctly folded and that, unlike the individual DBL domains, it binds with high affinity and specificity to CSA (K(D) = 61 nM) and efficiently inhibits PE from binding to CSA. Structural characterization by analytical ultracentrifugation and small-angle x-ray scattering reveals a compact organization of the full-length protein, most likely governed by specific interdomain interactions, rather than an extended structure. Collectively, these data suggest that a high-affinity, CSA-specific binding site is formed by the higher-order structure of the var2CSA extracellular region. These results have important consequences for the development of an effective vaccine and therapeutic inhibitors.
  Proceedings of the National Academy of Sciences 03/2010; 107(11):4884-9. · 9.68 Impact Factor
• Article: How does Plasmodium falciparum stick to CSA? Let's see in the crystal.

  Graham A Bentley, Benoît Gamain
  Nature Structural &#38 Molecular Biology 10/2008; 15(9):895-7. · 12.71 Impact Factor
• Article: Pregnancy-associated malaria: parasite binding, natural immunity and vaccine development.

  Benoît Gamain, Joseph D Smith, Nicola K Viebig, Jürg Gysin, Artur Scherf
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  ABSTRACT: Humans living in areas of high malaria transmission gradually acquire, during the early years of life, protective clinical immunity to Plasmodium falciparum, limiting serious complications of malaria to young children. However, pregnant women become more susceptible to severe P. falciparum infections during their first pregnancy. Pregnancy associated malaria is coupled with massive accumulation of parasitised erythrocytes and monocytes in the placental intervillous blood spaces, contributing to disease and death in pregnant women and developing infants. Indirect evidence suggests that prevention may be possible by vaccinating women of childbearing age before their first pregnancy. This review aims to introduce the reader to the implications of malaria infection during pregnancy and to analyse recent findings towards the identification and characterisation of parasite encoded erythrocyte surface proteins expressed in malaria-infected pregnant women that are likely targets of protective immunity and have potential for vaccine development.
  International Journal for Parasitology 04/2007; 37(3-4):273-83. · 3.39 Impact Factor