Production of Purified Recombinant Proteins for Development of Vaccines of Military Importance.

"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 therequirement 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 thishigh 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 forpre-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 candidateproteins 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 an