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Infection and Immunity, July 2006, p. 4357-4360, Vol. 74, No. 7
0019-9567/06/$08.00+0     doi:10.1128/IAI.01617-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Baculovirus-Expressed Constructs Induce Immunoglobulin G That Recognizes VAR2CSA on Plasmodium falciparum- Infected Erythrocytes

Lea Barfod, Morten A. Nielsen, Louise Turner, Madeleine Dahlbäck, Anja T. R. Jensen, Lars Hviid, Thor G. Theander, and Ali Salanti*

Centre for Medical Parasitology at Copenhagen University Hospital (Rigshospitalet) and Institute for Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark

Received 4 October 2005/ Returned for modification 3 January 2006/ Accepted 31 March 2006


   ABSTRACT
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We raised specific antisera against recombinant VAR2CSA domains produced in Escherichia coli and in insect cells. All were reactive in enzyme-linked immunosorbent assay, but only insect cell-derived constructs induced immunoglobulin G (IgG) that was reactive with native VAR2CSA on the surface of infected erythrocytes. Our data show that five of the six VAR2CSA Duffy-binding-like domains are surface exposed and that induction of surface-reactive VAR2CSA-specific IgG depends critically upon antigen conformation. These findings have implications for the development of vaccines against pregnancy-associated Plasmodium falciparum malaria.


   TEXT
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Pregnant women constitute an important exception to the general rule that malaria is concentrated among young children in areas with stable transmission of Plasmodium falciparum parasites (reviewed in reference 7). Pregnancy-associated malaria (PAM) is caused by parasites that selectively sequester in the placenta by means of particular parasite-encoded variant surface antigens (VSA) expressed on the surface of infected erythrocytes (IE). PAM-type VSA (VSAPAM) are functionally and antigenically distinct from all other VSA (1, 4, 12), explaining the pregnancy-associated reappearance of susceptibility to P. falciparum infection in previously immune women. Susceptibility to PAM rapidly decreases with increasing parity because of acquisition of VSAPAM-specific immunity (5, 18). The most important of the PAM-type VSA appears to be VAR2CSA, which is a member of the P. falciparum EMP1 protein family encoded by the multigene var family (13, 14). Levels of VAR2CSA-specific immunoglobulin G (IgG) are associated with acquired protection from PAM, and VAR2CSA is currently the leading candidate in PAM-specific vaccine development (13, 15).

Recombinant antigens and immunizations. The high predicted molecular mass of VAR2CSA (355 kDa) has frustrated determination of its structure and identification of surface-exposed domains that are accessible to antibodies and therefore are candidates for inclusion in a PAM-specific vaccine. To facilitate these studies, we sought to generate antisera reactive with native VAR2CSA on the surface of intact IE. We immunized BALB/c mice (eight times, 5 µg) and rabbits (eight times, 20 µg) with recombinant constructs corresponding to all six VAR2CSA Duffy-binding-like domains based on the var2csa sequence of P. falciparum clone 3D7 (PlasmoDB accession number PFL0030c) (6). Immuni-zation protocols were as described earlier (13). The VAR2CSA domains were cloned into PGEX-4T-1 (Amersham Biosciences, Hillerød, Denmark) for production of glutathione S-transferase-tagged recombinant proteins in Escherichia coli strains BL21 and BL21 Origami. The latter strain allows the formation of disulfide bonds that could lead to the production of biologically active protein that depend on such bonds for stabilization of tertiary structure (11). We also subcloned the var2csa domains into pBAD-TOPO vector (Invitrogen, Taastrup, Denmark), followed by cloning of plasmids into the baculovirus transfer vector pAcGP67-A (BD Biosciences, Brøndby, Denmark) and production of His-tagged recombinant proteins in baculovirus-infected Trichoplusia ni High-5 cells. We used both native domains and domains with codons changed for usage by T. ni (GenBank accession number DQ227316). However, as very little protein was produced when using native var2csa sequences, we abandoned this system. Using the VAR2CSA sequence with the codons changed, we obtained between 1 to 5 mg/liter insect cell. All recombinant proteins were purified and checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting as described previously (Fig. 1) (8).


Figure 1
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  		FIG. 1. VAR2CSA domain structure and characteristics of recombinant constructs. (A) Domain structure of VAR2CSA, with DBL domain boundaries indicated. (B and C) Positions of recombinant constructs produced as His-tagged proteins expressed in baculovirus-infected insect cells (B) or as glutathione S-transferase fusion proteins in E. coli (C). (D and E) Purity of constructs expressed in baculovirus-infected insect cells (D) or in E. coli (E) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

 
Analysis of recombinant antigens and antisera by ELISA. IgG with specificity for E. coli-produced and baculovirus-produced VAR2CSA domains was seen only in ELISA using plasma (1:100 dilution) from P. falciparum-exposed women living in Dodowa, Ghana (Fig. 2). In contrast, plasma from both men and women in Dodowa had similar plasma levels of IgG with specificity for the control antigen GLURP (Fig. 2A) (3). The selective sex-specific plasma IgG recognition pattern of the VAR2CSA constructs was reassuring, since it is a characteristic of VSAPAM, including VAR2CSA (12, 13). Antisera raised in animals immunized with each of the recombinant VAR2CSA domains reacted in ELISA exclusively with the domain used for vaccination (data not shown). The recombinant VAR2CSA thus had the human plasma IgG recognition pattern expected of VSAPAM constructs and elicited specific IgG responses in vaccinated animals.


Figure 2
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  		FIG. 2. Human plasma levels of VAR2CSA-specific and GLURP-specific IgG measured by ELISA. Median (central bar), central 50% (boxes), central 90% (whiskers), and outlier (circles) levels of human plasma IgG with specificity for recombinant constructs corresponding to all DBL domains of VAR2CSA produced in E. coli (A) or in baculovirus-infected High-5 cells (B) are shown. Reactivity with recombinant GLURP is shown for comparison in panel A. Plasma samples from Ghanaian P. falciparum-exposed men (M) (n = 26) and women (W) (n = 30) were used.

 
Analysis of recombinant antigens and antisera by flow cytometry. We next used flow cytometry to test the ability of the antisera to recognize native VAR2CSA on the surface of erythrocytes infected with NF54-VAR2CSA (17). This parasite line has been selected for high surface expression of VAR2CSA (13). None of the antisera raised against VAR2CSA domains produced in E. coli BL21 or BL21 Origami contained IgG reacting with the surface of NF54-VAR2CSA (Fig. 2 and data not shown). In contrast, antisera raised against all the baculovirus-produced domains except DBL4-{varepsilon} contained IgG reactive with the surface of intact IE (Fig. 3). These results show that most DBL domains in VAR2CSA are surface exposed and accessible to IgG in the native molecule as expressed on the surface of IE but that induction of such IgG depends critically upon the three-dimensional structure of the antigen used for immunization. The lack of IE surface reactivity in DBL4-{varepsilon} antisera (Fig. 3) despite their reactivity in ELISA (Table 1) likely indicates that the DBL4-{varepsilon} domain is not accessible to antibodies in correctly folded, native VAR2CSA. Recent studies of DBL domains in EBA-175 support this possibility (19). Finally, production of VAR2CSA vaccine constructs may require eukaryotic expression systems due to the apparent difficulties in obtaining correctly folded VAR2CSA constructs from E. coli.


Figure 3
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  		FIG. 3. Reactivity of domain-specific antisera with native VAR2CSA on the surface of P. falciparum-infected erythrocytes as measured by flow cytometry and immunofluorescence. Fluorescence of NF54-VAR2CSA-infected erythrocytes labeled either with VAR2CSA domain-specific murine antisera (open histograms) or with preimmunization control sera (shaded histograms) is shown. Adjuvants used were CpG (DBL1), Freund's adjuvant (DBL2, -3, -4, and -5), and alum (DBL6). The surface reactivities (by immunofluorescence microscopy) of corresponding rabbit antisera (with Freund's adjuvant) are shown as insets. Preimmunization sera and a corresponding antiserum with specificity for a non-VAR2CSA P. falciparum EMP1 (VAR4) (8) (with Freund's adjuvant) were negative by immunofluorescence microscopy (not shown).

 

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  		TABLE 1. Induction of VAR2CSA-specific IgG following immunization of mice and rabbits with recombinant domains and different adjuvants

 
Effect of adjuvants and immunization regimen. We used Freund's incomplete/complete adjuvant for immunization of both mice and rabbits. However, this adjuvant combination is not acceptable in humans, and we tested CpG DNA ImmunEasy (QIAGEN, Albertslund, Denmark), aluminum hydroxide gel (Sigma-Aldrich, Brøndby, Denmark), and immune stimulating complexes (kindly provided by Nikolai Kirkby, Rigshospitalet, Denmark) as alternative adjuvants in different groups of mice(reviewed in references 2, 9, 10, and 16). No clear pattern emerged from these experiments, as both CpG, Freund's adjuvant, and alum induced surface-reactive antibodies against three out of six domains and ISCOMS against two out of six (Table 1). Furthermore, DBL3-X and DBL5-{varepsilon} appeared to be intrinsically more immunogenic than the other domains, as these domains elicited surface-reactive IgG with a majority of the adjuvants. Levels of VAR2CSA-specific IgG detected by ELISA or by flow cytometry increased in parallel following immunization and peaked after five immunizations (data not shown).

Concluding remarks. We have shown that most domains of native VAR2CSA on the surface of intact IE are accessible to IgG and thus constitute potential vaccine candidates. We have also shown that adjuvants that are acceptable for use in humans can be used in vaccination protocols aimed at inducing such antibodies. Finally, this IgG appears to be directed mainly against conformational epitopes, a finding that has important implications for the development of antigens suitable for inclusion in PAM-specific vaccines.


   ACKNOWLEDGMENTS
 
We are indebted to the individuals who donated parasite and plasma samples for this study. We thank Anne Corfitz for excellent technical assistance.

This study received financial support from the Commission of the European Communities (grants QLK2-CT-2001-01302 [PAMVAC] and QLK2-CT-2002-01197 [EUROMALVAC2]), the Danish Medical Research Council (SSVF) (grants 22-02-0220 and 22-03-0333), and the Danish Research Council for Development Research (RUF) (grant 104.Dan.8.L.306). A.S. is supported by a postdoctoral grant from SSVF. L.B. is supported by a Ph.D. studentship from Hovedstadens Sygehusfaellesskab (Denmark). M.D. is supported by a Ph.D. studentship from RUF. M.A.N. is supported by a postdoctoral grant from Hovedstadens Sygehusfaellesskab.

A.S., A.T.R.J., L.H., M.D., and T.G.T. are owners of European patent WO2004067559 (http://ep.espacenet.com).


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute for Medical Microbiology and Immunology, Panum Institute 24-2, Blegdamsvej 3, 2200 Copenhagen N, Denmark. Phone: 45 35 32 76 76. Fax: 45 35 32 78 51. E-mail: salanti{at}cmp.dk. Back

Editor: J. F. Urban, Jr.


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Infection and Immunity, July 2006, p. 4357-4360, Vol. 74, No. 7
0019-9567/06/$08.00+0     doi:10.1128/IAI.01617-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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