Research Project
6443573
DESCRIPTION: (Provided by Applicant) 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. PROPOSED COMMERCIAL APPLICATION: There are an estimated 300-500 million new cases of malaria and 1.5-2.7 million deaths caused by malaria annually (World Health Organization, 1996). Every year an estimated tens of millions of North American, European, and Asian residents of non-malarious countries travel to countries where malaria is transmitted. They currently have to take antimalarial drugs which have side effects and are losing their effectiveness. In fact there are more than 1000 reported imported cases of malaria in the US each year, and it is estimated that several more thousand are not reported (Hoffman, 1992). Furthermore, during the 20th century when engaged in areas of the world where malaria is transmitted, the U.S. military has lost more person days to malaria than to bullets (Beadle and Hoffman, 1993). A vaccine against malaria would have enormous domestic and international commercial value.
