Pregnancy malaria (PM) affects estimated 50 million women worldwide and contributes to 200,000 infant deaths annually. Caused by sequestration of chondroitin sulfate A (CSA) binding Plasmodium falciparum parasites in placenta, PM increases risks of maternal anemia, stillbirth, spontaneous abortion, low birth weight, neonatal death, and preeclampsia. Previous ex vivo experiments have shown the mechanism of CSA binding in placenta can be inhibited by treating parasites with sera from multigravida women of malaria endemic area or animals immunized with antigens critical for parasite adhesion. LMIV is committed to develop pregnancy malaria vaccines that will protect women through production of anti-adhesion antibodies. During the past fiscal year, LMIV scientists have accomplished the following: 1. Continued development of two novel animal models of pregnancy malaria. Using a panel of P. chabaudi isolates (heterologous strains AS, CB and ER have been under study), we have developed a mouse model of both malaria recrudescence and malaria reinfection during pregnancy in C57BL6 mice, and have found that malaria severity and poor outcomes are related to production of inflammatory cytokines such as TNFa and MCP1, and the anti-inflammatory cytokine IL10. Our current pilot data suggest that P. chabaudi parasites may be sequestering in the mouse placenta, which would increase the relevance of this model to human placental malaria. Separately, we have developed the first ever non-human primate model of placental malaria, by showing the sequestration of P. falciparum parasites in the placenta of pregnant Aotus monkeys. Our data over the past year indicate that a single parasite clone causing infection during pregnancy can induce broadly neutralizing anti-adhesion antibodies in monkeysthis has important implications for vaccine development, suggesting that a single allele of varuiant antigen should be adequate to induce broadly active protection. 2. VAR2CSA has been the primary focus of PMV vaccine development for the malaria research community. In last years report, we described PfCSA-L as a novel surface antigen that associates with VAR2CSA on the surface knobs of Pf-IE. We have shown that PfCSA-L binds with high affinity to human placental CSPG by Surface Plasmon Resonance (SPR). Duo-Link analysis using DBL2X and PfCSA-L antibodies indicates that the proteins interact with each other;SPR analysis reveals that the DBL2X domain of VAR2CSA binds to PfCSA-L with subnanomolar affinity. Urea extraction of Pf-IE membranes suggests that both VAR2CSA and PfCSA-L are anchored by protein-protein (rather than protein-lipid) interactions on the IE surface, suggesting that they exist in complexes. Both VAR2CSA and PfCSA-L membrane proteins are resistant to alkaline sodium carbonate extraction, a hallmark of integral membrane proteins. These findings suggest that PfCSA-L interacts with VAR2CSA on surface knobs of Pf-IE, where it may contribute to the CSA-binding phenotype. As a highly conserved protein of small size (25 kDa), PfCSA-L appears to be a valuable target for placental malaria vaccine development. We are currently attempting to develop complexed forms of PfCSA-L and VAR2CSA so that we can study the protein complex as an immunogen. Our hypothesis is that the complex provides novel epitopes that are targets of broadly neutralizing antibodies. 3. Established new platforms to prepare candidate PMV candidates for animal studies, including baculovirus expression of recombinant protein, and preparation of DNA vaccines as a platform for relatively rapid assessment of similar immunogens (such as multiple constructs that examine alternative domain boundaries for the same VAR2CSA domain).
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