title: Var2CSA DBL6-epsilon domain expressed in HEK293 induces limited cross-reactive and blocking antibodies to CSA binding parasites.
abstract: BACKGROUND
  Pregnancy-associated malaria (PAM) is a serious consequence of Plasmodium falciparum-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.
  
  
  METHODS
  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 Escherichia coli 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-epsilon domains.
  
  
  RESULTS
  Using the HEK293 expression system, DBL1-X, DBL4-epsilon and DBL6-epsilon were produced at relatively high levels in the culture supernatant, while DBL3-X and DBL5-epsilon were produced at much lower levels. DBL2-X and DBL3-X domains were obtained after refolding of the inclusion bodies produced in E. coli. Importantly, mice antisera raised against the recombinant DBL6-epsilon domain, specifically reacted against the surface of CSA-binding parasites and revealed adhesion blocking activity.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institut Pasteur, Unité de Biologie des Interactions Hôte-Parasite, CNRS URA2581, Batiment Nicolle, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, France.
authors.forename: Pablo
authors.initials: P
authors.lastname: Fernandez
authors.forename: Nicola K
authors.initials: NK
authors.lastname: Viebig
authors.forename: Sébastien
authors.initials: S
authors.lastname: Dechavanne
authors.forename: Catherine
authors.initials: C
authors.lastname: Lépolard
authors.forename: Jürg
authors.initials: J
authors.lastname: Gysin
authors.forename: Artur
authors.initials: A
authors.lastname: Scherf
authors.forename: Benoit
authors.initials: B
authors.lastname: Gamain
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology*; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology*; D002448:Cell Adhesion / Q000276:immunology; D002460:Cell Line; D003429:Cross Reactions; D005260:Female; D006801:Humans; D008288:Malaria / Q000276:immunology / Q000517:prevention & control; D017780:Malaria Vaccines / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011247:Pregnancy; D011251:Pregnancy Complications, Infectious / Q000276:immunology / Q000517:prevention & control; D017434:Protein Structure, Tertiary
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D017780:Malaria Vaccines; C512601:VAR2CSA protein, Plasmodium falciparum
references.citation: Cell. 1995 Jul 14;82(1):77-87
references.pmid: 7541722
references.citation: Infect Immun. 2006 Jul;74(7):4357-60
references.pmid: 16790811
references.citation: Exp Parasitol. 2006 Feb;112(2):121-5
references.pmid: 16274691
references.citation: Nature. 1998 Oct 29;395(6705):851-2
references.pmid: 9804416
references.citation: Mol Microbiol. 2003 Jul;49(1):179-91
references.pmid: 12823820
references.citation: EMBO Rep. 2005 Aug;6(8):775-81
references.pmid: 16025132
references.citation: Cytometry. 1999 Apr 1;35(4):329-36
references.pmid: 10213198
references.citation: PLoS Med. 2006 Nov;3(11):e446
references.pmid: 17105340
references.citation: Placenta. 2004 May;25(5):359-78
references.pmid: 15081631
references.citation: J Immunol. 2000 Sep 15;165(6):3309-16
references.pmid: 10975848
references.citation: Infect Immun. 1999 Oct;67(10):5367-71
references.pmid: 10496918
references.citation: Am J Pathol. 1982 Dec;109(3):330-42
references.pmid: 6758604
references.citation: Mol Biochem Parasitol. 2006 Aug;148(2):117-24
references.pmid: 16631964
references.citation: Lancet. 2004 Jun 5;363(9424):1860-7
references.pmid: 15183624
references.citation: J Infect Dis. 1999 Aug;180(2):464-72
references.pmid: 10395863
references.citation: Exp Parasitol. 2005 Mar;109(3):195-7
references.pmid: 15713452
references.citation: J Immunol Methods. 1992 Jul 31;152(1):89-104
references.pmid: 1640112
references.citation: Int J Parasitol. 2007 Mar;37(3-4):273-83
references.pmid: 17224156
references.citation: Infect Immun. 2006 Aug;74(8):4875-83
references.pmid: 16861676
references.citation: Lancet. 2004 Jan 24;363(9405):283-9
references.pmid: 14751701
references.citation: J Infect Dis. 2005 Jul 15;192(2):331-5
references.pmid: 15962229
references.citation: Infect Immun. 2003 Nov;71(11):6620-3
references.pmid: 14573685
references.citation: Science. 1996 Jun 7;272(5267):1502-4
references.pmid: 8633247
references.citation: PLoS One. 2007 Sep 19;2(9):e910
references.pmid: 17878945
references.citation: Mol Med. 1997 Aug;3(8):508-18
references.pmid: 9307979
references.citation: J Infect Dis. 2006 Mar 1;193(5):713-20
references.pmid: 16453268
references.citation: J Exp Med. 2004 Nov 1;200(9):1197-203
references.pmid: 15520249
references.citation: Histopathology. 1993 Mar;22(3):211-8
references.pmid: 8495954
references.citation: J Infect Dis. 2005 Mar 15;191(6):1010-3
references.pmid: 15717280
references.citation: Science. 1976 Aug 20;193(4254):673-5
references.pmid: 781840
references.citation: Mol Biochem Parasitol. 2007 Oct;155(2):103-12
references.pmid: 17669514
references.citation: Cell. 1995 Jul 14;82(1):89-100
references.pmid: 7606788