FALCIPARUM MALARIA INVASION LIGAND DNA/PROTEIN VACCINE

The erythrocytic stage of Plasmodium falciparum (Pf) kills an estimated 2 million children annually. Pf invades erythrocytes (RBCs) by attaching to surface receptors, one of which includes sialic acids. We identified a novel erythrocyte binding protein-2 (EBP2) and the putative domain of the RBC receptor-binding ligand of Pf, the binding of which is sialic acid dependent. An EBP2 region II DNA vaccine induced antibodies that block EBP2 binding to RBCs. EBP2 is a paralogue of the well-characterized Pf 175 kDa erythrocyte binding protein (EBA-175) that binds RBCs and binding is sialic acid dependent. Preclinical immunogenicity studies in rabbits demonstrated that an EBA-175 region II protein alone or DNA prime/protein boost strategies could generate functional antibodies that block the binding of native EBA-175 and inhibit parasite invasion in vitro. An immunogenicity and efficacy study using this same EBA-175 region II DNA prime/protein boost strategy showed protection in Aotus monkeys. We have since enhanced the immunogenicity of the DNA vaccine and expressed EBA-175 region II in the methylotrophic yeast Pichia pastoris to high levels (100mg/L purified protein) using a synthetic EBA-175 region II gene fragment utilizing mammalian codons. We have experience with DNA vaccines and GMP experience with a high yield P. pastoris recombinant expression system. The specific aim is thus to construct a synthetic EBP2 region II gene fragment for construction of a DNA vaccine and recombinant protein expression in P. pastoris. Short term goals for recombinant EBP2 region II protein are: 1) fermentation and purification, 2) biochemical, and functional characterization, and 3) study generation of EBP2 blocking antibodies and efficacy to inhibit parasite growth in vitro. The long-term goal is large-scale production of a commercially viable EBP2 region II protein for preclinical Aotus safety, immunogenicity and efficacy studies alone and/or in combination with EBA-175 region II using a DNA prime/protein boost or protein alone vaccination regimen.