Expression, Purification And Efficacy testing of Synthetic Plasmodium Falciparum Apical Membrane Antigen 1 Expressed in Pichia Pastoris
The invention provides a synthetic gene encoding an allelic fonn of non-glycosylat~d Plasmodium falciparum Apical Membrane Antigen 1 optimized for codon usage in Pichia pastoris (e.g., AMAI FVa and AMAI 3D7); the subsequent transfonnation of Pichia pastoris with this gene, the separate optimization for pH, temperature, growth rate, and media composition of reproducible, scalable, clinical grade fennentation conditions for the expressionof the products of the gene; a method of purification for de-lipidation to clinical grade purity of the products of the gene; and proof of efficacy of the purified protein products in in vitro growth inhibition assays and in vivo non-human primate challenge models.
Expression, Purification And Efficacy testing of Synthetic Plasmodium Falciparum Apical Membrane Antigen 1 Expressed in Pichia Pastoris Two recombinant forms of the malaria asexual blood stage antigen Apical Membrane Antigen 1 AMA1 were produced in Pichia pastoris using totally defined, synthetic medias and a fermentation methodology that has been reproducibly scaled over a 10-fold range to 60L. High levels of secreted recombinant protein were obtained 300mg/L secreted protein in the supernatant, and >50mg/L final purified bulk protein , and a purification strategy developed to remove Host cell-derived lipids. Highly purified forms of both types of AMA1 produced appear to produce antibodies in vivo in rabbits that block homologous parasites from invading red blood cells in vitro. The combination of the two allelic forms made appears potent at inducing antibodies capable of blocking the invasion of many heterologous parasite strains in vitro, suggesting that the combination of these two alleles of AMA1 will provide sufficient coverage from the diverse field populations of parasites. One of the two AMA1's, based on the FVO allelic variant of AMA1, was emulsified with complete and incomplete Freund's adjuvant. Vaccination of highly susceptible Aotus vociferansmonkeys with this formulation conferred significant protection from a subsequent lethal challenge with the virulent FVO Plasmodium falciparumparasite. Five of eight animals whose primary immune response was directed against AMA1 were completely protected. These two recombinant forms of AMA1 may be an effective malaria vaccine. The production and purification methodologies may be suitable to other therapeutic proteins where large-scale, inexpensive production methodologies are required.
Abandoned Patent Reference Number(s): 60/344,752