Differential induction of functional IgG using the Plasmodium falciparum placental malaria vaccine candidate VAR2CSA

Abstract

Background: In Plasmodium falciparum malaria endemic areas placental malaria (PM) is an important complication of malaria. The recurrence of malaria in primigravidae women irrespective of acquired protection during childhood is caused by the interaction between the parasite-expressed VAR2CSA antigen and chondroitin sulfate A (CSA) in the placental intervillous space and lack of protective antibodies. PM impairs fetal development mainly by excessive inflammation processes. After infections during pregnancy women acquire immunity to PM conferred by antibodies against VAR2CSA. Ideally, a vaccine against PM will induce antibody-mediated immune responses that block the adhesion of infected erythrocytes (IE) in the placenta.

Principal findings: We have previously shown that antibodies raised in rat against individual domains of VAR2CSA can block IE binding to CSA. In this study we have immunized mice, rats and rabbits with each individual domain and the full-length protein corresponding to the FCR3 VAR2CSA variant. We found there is an inherently higher immunogenicity of C-terminal domains compared to N-terminally located domains. This was irrespective of whether antibodies were induced against single domains or the full-length protein. Species-specific antibody responses were also found, these were mainly directed against single domains and not the full-length VAR2CSA protein.

Conclusions/significance: Binding inhibitory antibodies appeared to be against conformational B-cell epitopes. Non-binding inhibitory antibodies reacted highly against the C-terminal end of the VAR2CSA molecule especially the highly polymorphic DBL6ε domain. Differential species-specific induction of antibody responses may allow for more direct analysis of functional versus non-functional B-cell epitopes.

Figure 1. Antibody responses to recombinant proteins in different species of animals.

The reactivity of VAR2CSA-specific IgG from mice (A), rats (B) and rabbits (C) immunized with individual domains (DBL1X-DBL6ε) and FV2 is shown as the mean of all animals immunized with a particular protein. Data are fitted to a sigmoid shaped dose response curve showing the different intrinsic antigenicity of the individual DBL-domains and FV2. For statistical analysis see Table S2.

Figure 2. Antibody reactivity against native and surface expressed VAR2CSA.

Serum from mice (A), rats (B) and rabbits (C) immunized with individual domains (DBL1X-DBL6ε) and FV2 was tested using FACS for reactivity against native VAR2CSA expressed on the surface of IE. Antibody surface reactivity is shown for FV2 and DBL1X-DBL6ε groups of animals as the median fluorescence intensity. Negative controls are serum from animals immunized with DBL3γVAR1 and a blank sample without serum.

Figure 3. Comparison of surface reactivity and inhibition of FCR3-IE adhesion to CSPG.

(A) Anti-VAR2CSA serum from rats and rabbits raised against DBL1X-DBL6ε and FV2 were tested using FCR3-IE for the ability of serum used in a 1∶10 dilution to inhibit binding to CSA in a 96-well plate assay. The data shown are the ratios of binding compared to the negative DBL3γVAR1 control ((Binding [test sample])/(Binding [negative control])). (B) IgG was purified from selected inhibitory and non-inhibitory sera and tested for reactivity in FACS at 1 mg/ml. DBL3γVAR1 was used as negative control. (C) Inhibition of binding to CSA using purified IgG at 0.5 mg/ml in the Petri dish assay. Error bars represents standard deviations.

Figure 4. Reactivity of FV2-specific antibodies to single recombinant DBL-domains.

The data shown are pools of serum from rats and rabbits immunized with FV2 tested in ELISA for reactivity towards single recombinant domains of VAR2CSA and FV2. Error bars represent standard deviations of triplicate measurements.

Figure 5. Antibody reactivity against DBL4ε epitopes measured by peptide analysis.

(A) Mean IgG reactivity against DBL4ε peptides in individual serum samples from rats (n = 10) (Dash dot line) and rabbits (n = 4) (Solid line) immunized with DBL4ε recombinant protein is measured using ELISA. Black bars represent epitopes mapped on the structure model of DBL4ε. (B) The structure model of the DBL4ε domain. The major peak (p3–p6) is yellow and the minor peak (p33–p38) is red.