Several domains from VAR2CSA can induce Plasmodium falciparum adhesion-blocking antibodies

Abstract

Background: Malaria caused by Plasmodium falciparum can result in several different syndromes with severe clinical consequences for the about 200 million individuals infected each year. During pregnancy, women living in endemic areas become susceptible to malaria due to lack of antibodies against a unique P. falciparum membrane protein, named VAR2CSA. This antigen is not expressed in childhood infections, since it binds chondroitin sulphate A (CSA) expressed on the intervillous space in the placenta. A vaccine appears possible because women acquire protective antibodies hindering sequestration in the placenta as a function of parity. A challenge for vaccine development is to design small constructs of this large antigen, which can induce broadly protective antibodies. It has previously been shown that one domain of VAR2CSA, DBL4-FCR3, induces parasite adhesion-blocking antibodies. In this study, it is demonstrated that other domains of VAR2CSA also can induce antibodies with inhibitory activity.

Methods: All VAR2CSA domains from the 3D7 and HB3 parasites were produced in Baculovirus-transfected insect cells. Groups of three rats per protein were immunized and anti-sera were tested for surface reactivity against infected erythrocytes expressing FCR3 VAR2CSA and for the ability to inhibit FCR3CSA parasite adhesion to CSA. The fine specificity of the immune sera was analysed by VAR2CSA peptide arrays.

Results: Inhibitory antibodies were induced by immunization with DBL3-HB3 T1 and DBL1-3D7. However, unlike the previously characterised DBL4-FCR3 response the inhibitory response against DBL1-3D7 and DBL3-HB3 T1 was poorly reproduced in the second rounds of immunizations.

Conclusion: It is possible to induce parasite adhesion-blocking antibodies when immunizing with a number of different VAR2CSA domains. This indicates that the CSA binding site in VAR2CSA is comprised of epitopes from different domains.

Figure 1

Surface reactivity and inhibitory capacity of serum specific for DBL domains of VAR2CSA from 3D7 and HB3. A and B: Sera from three rats immunized with the same recombinant protein were pooled and tested in FACS. The surface reactivity was measured using 10 μl serum in a total volume of 100 μl against 2 × 10⁵ late trophozoite and schizont-infected erythrocytes (IE). To measure the level of rat IgG bound to the surface of the IE 1 μl rabbit anti-rat IgG-FITC was used in a total volume of 100 μl and the mean fluorescence intensity of 5000 cells was analysed and shown in white bars. The assays were performed twice with similar results. C & D: For inhibition of IE binding to CSPG, 15 μl of rat serum in a total volume of 120 μl were tested in triplicate wells of 96 well Falcon plates coated with 2 μg/ml CSPG. Serum pools and tritium labelled parasites (2 × 10⁵) were added simultaneously to the wells and incubated for 2 hours. Hereafter the plate was washed using a pipetting robot and the remaining cells were harvested on a filter plate and counted using scintillation. The mean percentage of binding is shown relative to binding in wells without inhibitor. Error bars are standard deviations of triplicate measurements. The assays were performed twice with similar results. In both types of assays serum from rats immunized with FCR3 var1-DBL3γ and 3D7 PF08_0141-DBL2 β were used as negative controls.

Figure 2

Surface reactivity and inhibitory capacity of serum from rats immunized again with DBL3-HB3 T1 and DBL1-3D7. Surface reactivity (white bars) and inhibition of binding (black bars) assays were performed as described in Figure 1. White bars represent the ratio between the MFI of a given sample divided by MFI of the negative control. Black bars represent the ratio between the counts per minute (CPM) in a given sample divided by the CPM of the positive control. Error bars indicate standard deviations. A and B: Pools of sera from consecutive blood sampling from rats immunized with DBL3-HB3 T1. C Pools of sera from consecutive blood sampling from rats immunized with DBL1-3D7. The assays were performed twice with similar results.

Figure 3

Fine epitope mapping of anti-VAR2CSA IgG. The fine specificity of the antibody response in inhibitory and non-inhibitory sera was analysed using a peptide array covering the extracellular part of 3D7 VAR2CSA. Inhibitory rat sera are shown with red vertical bars and non-inhibitory sera with black or blue vertical bars. IgG from three individual rats immunized with DBL1-3D7 (A) and DBL3-HB3 T1 (B) were analysed and IgG from two individual rats immunized with DBL4-FCR3 (red bars) and DBL4-3D7 (black bars) (C). B-cell epitopes are marked on the graph with horizontal bars in blue, red or orange and were mapped on a structural model of the corresponding domain based on the 3D7 sequence.