Lyophilization of P. falciparum Transgenic for Trehalose Synthesis/Transport

描述（由申请人提供）：最近呼吁消除恶性疟原虫（Pf）引起的疟疾，并最终根除所有疟疾，这一疾病引起了人们的关注，它每年造成数亿病例和100万人死亡。消灭Pf的理想工具是一种非常有效的疫苗，它可以防止血液阶段感染，从而防止所有疾病和传播。Pf是造成99%疟疾死亡的病原体。当被受感染的蚊子叮咬给予减毒的Pf孢子虫（PfSPZ）时，b> 90%的人类志愿者受到保护，免受实验性Pf攻击，保护持续至少10个月。Sanaria的目标是开发并商业化一种减毒PfSPZ疫苗，该疫苗可在90%的受体中预防Pf血期感染；一种可以从世界上消灭Pf病毒的疫苗。这种疫苗有潜力在发达国家和发展中国家的市场上每年创造10亿美元的收入。Sanaria已经成功地建立了稳健的、可重复的、一致的PfSPZ疫苗的生产和临床批次的发布，获得了FDA的批准进行临床试验（IND批准），并于2009年5月启动了一项1期临床试验，以评估PfSPZ疫苗的安全性、免疫原性和保护功效。在该试验中证明了安全性之后，目标是尽快在非洲成人、幼儿和婴儿中进行安全性和概念有效性验证研究。随后将进行额外的2期研究，包括剂量优化研究，然后是关键的3期研究，以支持许可。在目前的配方中，PfSPZ疫苗储存在液氮气相中，温度低于-140℃。开发一种不需要液氮的储存方案可以大大简化其交付的物流，并使PfSPZ疫苗能够适应现有的疫苗分配系统。冻干是目前在许多行业中使用的一种热稳定方法，它依靠从生物样品中去除细胞内的水来防止随着时间的推移而降解。在初步研究中，我们已经朝着使用几种方法干燥PfSPZ的目标取得了进展，但还不可能生产出在环境温度下（4℃或-20℃）比组成我们的PfSPZ疫苗更稳定的寄生虫。我们对细胞外和细胞内配方进行了实验，这些配方在其他系统中已被证明具有冻干保护作用，并得出结论，PfSPZ的成功冻干或干燥需要PfSPZ内部大量存在冻干保护剂。最有效的冻干保护剂是海藻糖，它自然存在于耐干燥的生物体中。我们无法使用多种方法来内化海藻糖，包括电穿孔、DMSO渗透和热冲击，并得出结论，海藻糖的内化将需要PfSPZ自己生产或进口这种糖。本研究的目标是克隆负责海藻糖合成或海藻糖摄取的外源基因到PfSPZ中，以便这些基因产物的表达将赋予PfSPZ对干燥的耐受性，并使其在干燥或冻干状态下保持活力和效力，从而提供成功的热稳定PfSPZ疫苗。

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.