Genetic diversity of VAR2CSA ID1-DBL2Xb in worldwide Plasmodium falciparum populations: impact on vaccine design for placental malaria.

In placental malaria (PM), sequestration of infected erythrocytes in the placenta is mediated by an interaction between VAR2CSA, a Plasmodium falciparum protein expressed on erythrocytes, and chondroitin sulfate A (CSA) on syncytiotrophoblasts. Recent works have identified ID1-DBL2Xb as the minimal CSA-binding region within VAR2CSA able to induce strong protective immunity, making it the leading candidate for the development of a vaccine against PM. Assessing the existence of population differences in the distribution of ID1-DBL2Xb polymorphisms is of paramount importance to determine whether geographic diversity must be considered when designing a candidate vaccine based on this fragment. In this study, we examined patterns of sequence variation of ID1-DBL2Xb in a large collection of P. falciparum field isolates (n=247) from different malaria-endemic areas, including Africa (Benin, Senegal, Cameroon and Madagascar), Asia (Cambodia), Oceania (Papua New Guinea), and Latin America (Peru). Detection of variants and estimation of their allele frequencies were performed using next-generation sequencing of DNA pools. A considerable amount of variation was detected along the whole gene segment, suggesting that several allelic variants may need to be included in a candidate vaccine to achieve broad population coverage. However, most sequence variants were common and extensively shared among worldwide parasite populations, demonstrating long term persistence of those polymorphisms, probably maintained through balancing selection. Therefore, a vaccine mixture including such stable antigen variants will be putatively applicable and efficacious in all world regions where malaria occurs. Despite similarity in ID1-DBL2Xb allele repertoire across geographic areas, several peaks of strong population differentiation were observed at specific polymorphic loci, pointing out putative targets of humoral immunity subject to positive immune selection.

Identification of Id1-DBL2X of VAR2CSA as a key domain inducing highly inhibitory and cross-reactive antibodies.

PURPOSE OF THE RESEARCH: VAR2CSA is considered as the main target of protective immunity against pregnancy-associated malaria. VAR2CSA high molecular weight complicates scaling up production of VAR2CSA recombinant protein for large-scale vaccination programmes. We previously demonstrated that antibodies induced by NTS-DBL1X-Id1-DBL2X efficiently block parasite binding to CSA in a similar manner to antibodies induced by the full-length extracellular part of VAR2CSA. In order to identifying the shortest fragment of VAR2CSA carrying major protective epitopes able to elicit inhibitory antibodies, we performed a refined antigenic mapping of NTS-DBL1X-Id1-DBL2X through a DNA vaccination technique. PRINCIPAL RESULTS: Five single or double domains constructs encoding NTS-DBL1X, NTS-DBL1X-Id1, Id1, Id1-DBL2X and DBL2X were made and used to immunize mice. The NTS-DBL1X, NTS-DBL1X-Id1, and Id1-DBL2X fragments all raised high titer immune response, as measured by ELISA. The DBL2X fragment raised a weaker antibody titer, and the Id1 construct failed to elicit antibody. Sera from mice immunized with NTS-DBL1X or DBL2X constructs failed to block infected erythrocytes binding to CSA, whereas sera from mice immunized with NTS-DBL1X-Id1 showed partial inhibitory activity, and the Id1-DBL2X fragment elicited antisera that totally abrogated infected erythrocytes adhesion to CSA. IgG purified from Id1-DBL2X antisera showed a similar inhibitory profile than Id1-DBL2X antisera. Anti-FCR3 anti-Id1-DBL2X antibodies also efficiently block the adhesion of erythrocytes infected by the HB3 parasite line to CSA. Id1-DBL2X antisera recognized the surface of field isolates from pregnant women, and inhibited CSA-binding of all 8 isolates tested, although to a variable level. MAJOR CONCLUSIONS: We raised high-titer antibodies against several parts of the protein, and identified Id1-DBL2X as the minimal VAR2CSA fragment inducing antibodies with CSA-binding inhibitory efficiency in the same range as the full-length extracellular part of VAR2CSA.

Electroporation-mediated genetic vaccination for antigen mapping: application to Plasmodium falciparum VAR2CSA protein.

Genetic vaccination, consisting in delivering a genetically engineered plasmid DNA by a non-viral vector or technique into a tissue, is currently of great interest. New delivery technique including DNA transfer by electroporation recently greatly improved the potency of this concept. Because it avoids the step of producing a recombinant protein, it is particularly of use in studying the immunogenic properties of large proteins. Here we describe the use of electroporation mediated DNA immunization to identify important protective epitopes from the large VAR2CSA protein from Plasmodium falciparum implicated in the pathology of placental malaria. Immunizing mice and rabbit with DNA plasmids encoding different fragments of VAR2CSA leads to high titer antisera. Moreover an N-terminal region of the protein was found to induce protective functional antibodies.