Baculovirus-expressed constructs induce immunoglobulin G that recognizes VAR2CSA on Plasmodium falciparum-infected erythrocytes

Abstract

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.

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.

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.

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).