Lyophilization of P. falciparum Transgenic for Trehalose Synthesis/Transport

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DESCRIPTION (provided by applicant): The recent call for elimination of malaria caused by Plasmodium falciparum (Pf) and eventual eradication of all malaria has focused attention on this disease, which is responsible for hundreds of millions of cases and a million deaths annually. An ideal tool for eliminating Pf, the causative agent of 99% of all malaria deaths, would be a highly effective vaccine that prevents blood stage infection and thereby prevents all disease and transmission. When attenuated Pf sporozoites (PfSPZ) are administered by the bite of infected mosquitoes, > 90% of human volunteers are protected against experimental Pf challenge and protection lasts at least 10 months. Sanaria's goal is to develop and commercialize an attenuated PfSPZ vaccine that prevents Pf blood stage infection in > 90% of recipients; a vaccine that could be used to eliminate Pf from the world. This vaccine has the potential for > $1 billion annual revenues in markets in the developed and developing world. Sanaria has succeeded in establishing robust, reproducible, and consistent manufacture and release of clinical lots of its PfSPZ Vaccine, received FDA clearance to proceed with clinical trials (IND approval), and in May 2009 initiated a Phase 1 clinical trial to assess safety, immunogenicity, and protective efficacy of the PfSPZ Vaccine. After demonstrating safety in that trial, the goal is to move as swiftly as possible to safety and proof of concept efficacy studies in African adults, young children, and infants. These will be followed by additional Phase 2 studies, including dose optimization studies, and then pivotal Phase 3 studies to support licensure. In its current formulation, the PfSPZ Vaccine is stored in liquid nitrogen vapor phase at temperatures below -140¿C. Developing a storage protocol that does not require liquid nitrogen could considerably ease the logistics of its delivery and enable the PfSPZ Vaccine to fit into the existing vaccine distribution system. Lyophilization, a thermostabilization method currently used in many industries, relies on removal of intracellular water from biological samples to protect against degradation over time. In preliminary studies, we have made progress toward the goal of drying PfSPZ using several methods, but it has not yet been possible to produce a parasite that is more stable at ambient temperature, 4¿C, or -20¿C than those that comprise our PfSPZ Vaccine. We have experimented with extracellular and intracellular formulations that have been shown to be lyoprotective in other systems, and have concluded that the successful lyophilization or desiccation of PfSPZ requires the internal presence of lyoprotectants in quantity in the PfSPZ. The most effective lyoprotectant, which occurs naturally in desiccation-tolerant organisms, is the disaccharide trehalose. We have not been able to internalize trehalose using multiple methods, including electroporation, DMSO permeabilization, and heat shock, and have concluded that internalization of trehalose will require the PfSPZ to produce or to import this sugar themselves. The goal of this proposal is to clone exogenous genes responsible for trehalose synthesis or trehalose uptake into PfSPZ so that the expression of the products of these genes will impart desiccation tolerance on PfSPZ and allow their viability and potency to be retained in the desiccated or lyophilized state and provide a successful alternatively thermostabilized PfSPZ vaccine. PUBLIC HEALTH RELEVANCE: Malaria causes 500 million clinical cases and 1-3 million deaths annually, is responsible for >1% loss of GDP in Africa annually and is a serious concern for travelers and military personnel; Sanaria's goal is to develop and commercialize a >90% protective malaria vaccine for three primary markets with a potential for >$1 billion annual revenues. Though we have demonstrated that a frozen live, attenuated parasite vaccine has unprecedented protective efficacy and potential, a dried formulation that does not require refrigeration would allow the vaccine to be more easily produced, transported, and stored. Since the parasites in the current vaccine formulation cannot withstand the freeze drying process, we propose a genetic manipulation that will permit the parasites to generate or import a sugar that will enhance their own survival under drying conditions.