Lyophilization of P. falciparum Transgenic for Trehalose Synthesis/Transport

描述(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，将注意力集中在这种疾病上，这种疾病每年造成数亿病例和100万人死亡。消除肺泡炎的理想工具是一种高效疫苗，它可以预防血液阶段感染，从而防止所有疾病和传播。肺结核病是导致99%的疟疾死亡的原因。当减毒肺泡子孢子虫(PfSPZ)通过感染蚊子的叮咬给药时，90%的人类志愿者对实验性肺炎的攻击具有保护作用，保护持续至少10个月。Sanaria的目标是开发一种减毒PfSPZ疫苗并将其商业化，以防止90%的接受者发生肺泡炎血液期感染；这是一种可用于从世界上消除肺炎的疫苗。这种疫苗在发达国家和发展中国家的市场上有可能带来10亿美元的年收入。Sanaria已经成功地建立了其PfSPZ疫苗的强健、可重复和一致的临床批量生产和释放，获得了FDA的批准，可以继续进行临床试验(IND批准)，并于2009年5月启动了一项第一阶段临床试验，以评估PfSPZ疫苗的安全性、免疫原性和保护效果。在该试验中证明了安全性之后，目标是尽可能快地在非洲成年人、幼儿和婴儿中进行安全性和概念验证疗效研究。随后将进行额外的第二阶段研究，包括剂量优化研究，然后是支持许可的关键第三阶段研究。在目前的配方中，PfSPZ疫苗在-140℃以下的液氮蒸气相中储存。开发一种不需要液氮的储存方案可以极大地简化其输送的物流，并使PfSPZ疫苗能够适应现有的疫苗分配系统。冷冻干燥是目前在许多行业中使用的一种热稳定方法，它依赖于从生物样品中去除细胞内的水分，以防止随着时间的推移而降解。在初步研究中，我们已经使用几种方法朝着干燥PfSPZ的目标取得了进展，但还不可能产生一种在常温下比我们的PfSPZ疫苗更稳定的寄生虫。我们已经对在其他系统中被证明具有溶血保护作用的细胞外和细胞内制剂进行了实验，并得出结论，PfSPZ的成功冷冻干燥或干燥需要在PfSPZ中存在大量的体内溶血保护剂。天然存在于耐脱水生物体中的最有效的凝胶保护剂是二糖海藻糖。我们还不能使用多种方法内化海藻糖，包括电穿孔、DMSO渗透和热休克，并得出结论，内化海藻糖将需要PfSPZ自己生产或进口这种糖。这项建议的目的是将负责海藻糖合成或海藻糖吸收的外源基因克隆到PfSPZ中，以便这些基因的产物的表达将赋予PfSPZ对干燥的耐受性，并允许它们在干燥或冷冻干燥状态下保持其活性和效力，并提供一种成功的替代热稳定性PfSPZ疫苗。 与公共卫生相关：疟疾每年导致5亿临床病例和100-300万人死亡，每年造成非洲国内生产总值1%的损失，是旅行者和军事人员严重关切的问题；萨那里亚的目标是为三个初级市场开发和商业化90%保护性疟疾疫苗，年收入可能达到10亿美元。尽管我们已经证明了冷冻的减毒活寄生虫疫苗具有前所未有的保护效力和潜力，但不需要冷藏的干燥制剂将使疫苗更容易生产、运输和储存。由于目前疫苗配方中的寄生虫无法承受冷冻干燥过程，我们提出了一种基因操作，允许寄生虫产生或输入一种糖，从而提高它们在干燥条件下的存活率。

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.