SBIR-STTR Award

Production of malaria proteins in recombinant protein expression systems in a commercially viable manner for use in vaccine development and in in vitro assays has been complicated by the adenine/thymine rich genome of Plasmodium falciparum and the requirement for properly folded recombinant protein. To overcome both of these constraints, we propose to alter the codon usage of P. falciparum genes for expression in the eukaryotic methylotrophic yeast Pichia pastoris. We have GMP experience with this high yield P. pastoris recombinant expression system for manufacture of therapeutic proteins and experience producing Region II of the P. falciparum erythrocyte binding protein (EBA-175) in P. pastoris at quantities of 125 mg/L fermentation supernatant for pre-clinical malaria vaccine development. The specific aim is thus to express P. falciparum proteins in P. pastoris. Short term goals for recombinant protein production are: 1) expression and purification, 2) biochemical, and functional characterization, and 3) study immunogenicity in mice. The Phase II goals are optimization of expression and purification, and analysis of protective efficacy in a non-human primate model, and in Phase III the goals are large-scale GMP production of vaccine candidate proteins for Phase I/II human studies alone and/or in combination with a DNA vaccine. 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.

Malaria caused by Plasmodium falciparum affects 300-500 million and kills 1-3 million individuals annually. For more than 150 years during every military campaign conducted where malaria was transmitted, U.S. forces have had more casualties from malaria than from hostile fire. For these reasons malaria vaccine development is one of the highest ranked objectives for DoD biomedical R&D. Five P. falciparum proteins, PfCSP, PfSSP2, PfLSA1, PfAMA1 and PfMSP1 are considered prime targets for inclusion in a malaria vaccine. In a Phase I SBIR we demonstrated the feasibility of producing one of these proteins, PfCSP, in a highly immunogenic form. In Phase II we will produce 20-50 mg of purified, well characterized, recombinant proteins based on these antigens, demonstrate that the proteins are immunogenic in mice, and provide the proteins to the Naval Medical Research Center for use as reagents in their vaccine development program. In addition we will produce protocols and reagents that are transferable to Investigational New Drug (IND) applications and FDA directed studies. Thus, by the end of Phase II, we will have the data, material, and protocols required to take these proteins forward, not only as reagents, but also as vaccines on their own

Keywords:
Plasmodium Falciparum Recombinant Proteins, Plasmodium Falciparum Vaccines, Malaria Vaccines, Malaria, Military Vaccines, Production And Process Devel