Human pregnancy-associated malaria-specific B cells target polymorphic, conformational epitopes in VAR2CSA

Abstract

Pregnancy-associated malaria (PAM) is caused by Plasmodium falciparum-infected erythrocytes (IEs) that bind to chondroitin sulphate A (CSA) in the placenta by PAM-associated clonally variant surface antigens (VSA). Pregnancy-specific VSA (VSA(PAM)), which include the PfEMP1 variant VAR2CSA, are targets of IgG-mediated protective immunity to PAM. Here, we report an investigation of the specificity of naturally acquired immunity to PAM, using eight human monoclonal IgG1 antibodies that react exclusively with intact CSA-adhering IEs expressing VSA(PAM). Four reacted in Western blotting with high-molecular-weight (> 200 kDa) proteins, while seven reacted with either the DBL3-X or the DBL5-epsilon domains of VAR2CSA expressed either as Baculovirus constructs or on the surface of transfected Jurkat cells. We used a panel of recombinant antigens representing DBL3-X domains from P. falciparum field isolates to evaluate B-cell epitope diversity among parasite isolates, and identified the binding site of one monoclonal antibody using a chimeric DBL3-X construct. Our findings show that there is a high-frequency memory response to VSA(PAM), indicating that VAR2CSA is a primary target of naturally acquired PAM-specific protective immunity, and demonstrate the value of human monoclonal antibodies and conformationally intact recombinant antigens in VSA characterization.

Fig. 1

Flow cytometry analysis of human VSA-specific plasma IgG reactivity with the surface of P. falciparum-IEs. Labelling of FCR3-CSA, NF54-VAR2CSA and 3D7-SM by IgG in individual plasma samples from P. falciparum-exposed pregnant women (▴), from sympatric men (▾) and from non-exposed adult control donors (•) are shown.

Fig. 2

Reactivity of human IgG monoclonal antibodies with P. falciparum-IEs. A. Labelling of FCR3-CSA, NF54-VAR2CSA and 3D7-SM by VSA_PAM-specific monoclonal antibodies (•) or an irrelevant control monoclonal antibody (–), determined by flow cytometry. B. Reactivity of monoclonal antibody PAM3.10 (heavy line) and an irrelevant control monoclonal antibody (thin line) with the surface of erythrocytes infected by FCR3-CSA. C. PAM3.10 reactivity with the surface of erythrocytes infected by unselected FCR3. D. Immunofluorescence microscopy of FCR3-CSA-infected erythrocytes labelled with PAM3.10 (top) or an irrelevant control antibody (bottom). E. Immunofluorescence microscopy of unselected FCR3-IEs labelled with PAM3.10 (top) or an irrelevant control antibody (bottom). F. Reactivity of PAM3.10 in Western blots of FCR3-CSA (left) and FCR3 (centre). Broad-range molecular weight markers are shown in the right lane.

Fig. 3

Reactivity of human VSA_PAM-specific IgG1 monoclonal antibodies with VAR2CSA. A. The top panel shows a schematic representation of VAR2CSA with the positions of the recombinant protein constructs used (A–Z) and the amino acid numbers indicated along the bottom. Recognition of constructs A, E, I, L, P, T and Z in ELISA, and flow cytometric recognition of Jurkat cells transfected to express constructs B–D, F–H, J, K, M–O, Q–S, U–Y by antibodies PAM2.8 (red), PAM3.10 (orange), PAM5.2 and PAM7.5 (green), and PAM6.1 (blue) are shown. B. PAM6.1-specific competition ELISA to determine domain specificity of DBL3-X-reactive IgG (competitors shown in the figure). C. PAM3.10-specific competition ELISA to determine domain specificity of DBL5-ε-reactive IgG (competitors shown in the figure).

Fig. 4

PAM8.1 recognition of VAR2CSA DBL3-X. A. Amino acid sequence in the region of the domain where interclonal variation affected PAM8.1 recognition of Baculovirus-produced DBL3-X constructs from 29 genetically distinct P. falciparum isolates, including the sequence of a chimeric protein constructed to add PAM8.1 reactivity to the otherwise PAM8.1-negative 3D7 VAR2CSA DBL3-X sequence. B. Structural model of the 3D7 DBL3-X domain. The predicted loop region where parasite isolates recognized by PAM8.1 have a definite insertion compared with 3D7 is shown in red. The 3D7 residues flanking the insert, G1474 and Q1475 (positions 26 and 39 in A), are highlighted in black. C. Western blots of recombinant 3D7- and FCR3-specific VAR2CSA DBL3-X constructs, and of the above-mentioned chimeric construct, probed with loading control antibody V5 (left) and PAM8.1 (right). MW, molecular weight.

Fig. 5

PAM1.4 selection of parasite line EJ27. A. Pre-selection reactivity of monoclonal antibody PAM1.4 (heavy line) and negative control monoclonal antibody (thin line) with the surface of EJ27-IEs. B. Pre-selection non-PAM VSA-type recognition pattern of EJ27 by IgG in plasma from P. falciparum-exposed men and women and in plasma from non-exposed adults. C. Reactivity of PAM1.4 antibody (heavy line) and negative control antibody (thin line) with the surface of erythrocytes infected by the EJ27 after a single round of selection for reactivity with PAM1.4. D. Post-selection VSA_PAM-type recognition pattern of EJ27 by IgG in plasma from P. falciparum-exposed men and women and in plasma from non-exposed adults.