Immunization with VAR2CSA-DBL5 recombinant protein elicits broadly cross-reactive antibodies to placental Plasmodium falciparum-infected erythrocytes

Abstract

Pregnancy-associated malaria is a severe clinical syndrome associated with the sequestration of Plasmodium falciparum-infected erythrocytes in the placenta. Placental binding is mediated by VAR2CSA, a member of the large and diverse P. falciparum erythrocyte membrane 1 (PfEMP1) protein family. To better understand if conserved regions in VAR2CSA can be targeted by antibodies, we immunized rabbits with VAR2CSA-DBL1 and -DBL5 recombinant proteins produced in Pichia pastoris and developed a panel of seven chondroitin sulfate A (CSA)-binding parasites from diverse geographic origins. Overall, no two parasites in the panel expressed the same VAR2CSA sequence. The DBL1 domains averaged 80% amino acid identity (range, 72 to 89%), and the DBL5 domains averaged 86% amino acid identity (range, 83 to 99%), similar to a broader sampling of VAR2CSA sequences from around the world. Whereas antibodies generated against the VAR2CSA-DBL1 recombinant protein had only limited breadth and reacted with three or four parasites in the panel, immunization with DBL5 recombinant proteins elicited broadly cross-reactive antibodies against all or most parasites in the panel, as well as to fresh clinical isolates from pregnant women. These findings demonstrate that the major PfEMP1 variant expressed by placental isolates exposes strain-transcendent epitopes that can be targeted by vaccination and may have application for pregnancy malaria vaccine development.

FIG. 1.

Expression of VAR2CSA-DBL5 recombinant proteins in P. pastoris. (A) Protein schematic of VAR2CSA. The original DBL domain boundaries are numbered and indicated by black rectangles. In gray are new, longer domain boundaries predicted by X-ray crystallographic analysis of DBL disulfide bonds (27, 30). The first and last amino acids in VAR2CSA constructs are indicated below the protein schematic. The arrowhead indicates that the 3D7-DBL5 protein was truncated in the N terminus at the amino acid position 95, which resulted in a loss of ∼10 kDa of the protein. One microgram of recombinant proteins was analyzed under nonreducing conditions in a 4 to 20% SDS-PAGE gel and stained by GelCode blue reagent (B) or detected by Western blotting using anti-His tag antibodies (C). MW, molecular weight.

FIG. 2.

Sequence comparison of VAR2CSA-DBL5. Neighbor-joining trees comparing DBL1 (A) and DBL5 (B) sequences from CSA-binding parasites in the panel to global isolates. Bootstrap values above 80% are shown. The geographic origin of parasites is indicated by color: yellow, Southeast Asia; light brown, Africa; metallic brown, Central or South America; gray, unknown. Parasites in the reference panel are indicated by oval-shaped or rectangular shading. DBL5 recombinant proteins were made from parasites with rectangular shading.