Lyophilization of Plasmodium falciparum sporozoite vaccine

疟疾每年造成5亿例临床病例和1-3百万例死亡，每年造成非洲GDP损失>1%，是旅行者和军事人员的严重关切。当减毒的恶性疟原虫子孢子（PfSPZ）通过感染的蚊子的叮咬施用时，>90%的人类志愿者被保护免于实验性Pf攻击，并且保护持续至少10.5个月。Sanaria的目标是为3个市场开发和商业化>90%的保护性减毒PfSPZ疫苗，年收入可能> 10亿美元：1）发达国家的旅行者，2）发展中国家的婴儿和幼儿，以及3）发展中国家的青春期女孩。在单独的II期SBIR中，我们正在开发原型，PfSPZ疫苗，其使用NF 54 Pf菌株，使用冷冻保存作为活减毒子孢子的储存方法。冷冻保存协议产生高产量的可行PfSPZ下的气相氮储存在低于-135？C.利用冻干的储存方案的开发将对疫苗的储存及其递送的物流产生相当大的益处。冻干是一种通常用于储存非活疫苗的方法，并且其也常规用于储存许多原核生物以及某些原生动物的储备菌株。在初步研究中，PfSPZ耐受冻干保护剂良好，包括通常用于其他商业注射产品冻干的那些试剂，并且PfSPZ可以使用这些化合物作为冷冻保护剂成功地冷冻保存。本提案的目的是开发一种用于减毒PfSPZ活疫苗的冻干方法，该方法可扩大规模用于减毒PfSPZ活疫苗的大规模生产、储存和分销。我们用于验证和定量原型减毒PfSPZ疫苗效力的体外试验将用于证明冻干PfSPZ是可行的。这是通过证明冻干和复溶的PfSPZ能够侵入人肝细胞并在肝细胞中表达在子孢子中不表达的蛋白质来完成的。在II期SBIR和后续研究中，我们将把储存减毒PfSPz活疫苗的冻干方法纳入疫苗的cGMP生产工艺，并在临床试验中评估冻干PfSPZ疫苗的安全性、免疫原性和保护效力。疟疾每年造成5亿临床病例和1-3百万人死亡，每年造成非洲GDP损失>1%，是旅行者和军事人员的严重关切。Sanaria的目标是为主要市场开发和商业化一种保护性>90%的疟疾疫苗，年收入可能超过10亿美元; 1）来自发达国家的旅行者，2）发展中国家的婴儿，幼儿和少女。一种有效的恶性疟原虫疟疾疫苗将从根本上改变贫穷和疾病的循环，并将加快对非洲和其他受影响地区的经济投资。我们已经证明，冷冻减毒寄生虫疫苗具有前所未有的保护效力和潜力。然而，已经干燥且不需要冷藏的疫苗将使疫苗更容易生产、运输和储存。干疫苗有望降低疫苗成本，这是为高危人群生产数百万剂疫苗时的一个重要经济因素。此外，干疫苗可能会加速世界范围内消除这种毁灭性疾病的协调努力。

DESCRIPTION (provided by applicant): Malaria causes 500 million clinical cases and 1-3 million deaths annually, is responsible for a loss of >1% of GDP in Africa annually, and is a serious concern for travelers and military personnel. When attenuated Plasmodium falciparum sporozoites (PfSPZ) are administered by bite of infected mosquitoes >90% of human volunteers are protected against experimental Pf challenge and protection lasts at least 10.5 months. Sanaria's goal is to develop and commercialize a >90% protective attenuated PfSPZ vaccine for 3 markets with a potential for >$1 billion annual revenues; 1) Travelers from the developed world, 2) Infants and young children in the developing world, and 3) Adolescent girls in the developing world. In a separate Phase II SBIR, we are developing the prototype, a PfSPZ vaccine using the NF54 strain of Pf using cryopreservation as the method of storage of the live attenuated sporozoites. The cryopreservation protocol produces high yields of viable PfSPZ which are banked under vapor phase nitrogen storage at below -135?C. The development of a storage protocol that utilizes lyophilization would produce a considerable benefit to storage of the vaccine and to the logistics of its delivery. Lyophilization is a process commonly used to store non-living vaccines, and which is also in routine use for the storage of stock strains of many prokaryotes and also certain protozoa. In preliminary studies, PfSPZ tolerate lyoprotective agents well, including those agents commonly used in the lyophilization of other commercial injectable products, and PfSPZ can be cryopreserved successfully using these compounds as cryoprotective agents. The goal of this proposal is to develop a lyophilization method for live attenuated PfSPZ that can be scaled up for large scale manufacturing, storage and distribution of the live attenuated PfSPZ vaccine. The in vitro assay that we use to validate and quantify the potency of the prototype attenuated PfSPZ vaccine will be used to demonstrate that lyophilized PfSPZ are viable. This is done by demonstrating that the lyophilized and reconstituted PfSPZ are able to invade human hepatocytes and express proteins in the hepatocytes that are not expressed in sporozoites. In a Phase II SBIR and subsequent studies, we will incorporate the lyophilization method for the storage of live, attenuated PfSPz into the cGMP manufacturing process for the vaccine, and assess the safety, immunogenicity and protective efficacy of the lyophilized PfSPZ vaccine in clinical trials. 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 primary markets with a potential for >$1 billion annual revenues; 1) Travelers from the developed world, and 2) Infants, young children, and adolescent girls in the developing world. An effective vaccine against Plasmodium falciparum malaria will fundamentally alter the cycle of poverty and illness and will accelerate economic investment into Africa and other afflicted areas. We have demonstrated that a frozen attenuated-parasite vaccine has unprecedented protective efficacy and potential. However, a vaccine that has been dried and does not require refrigeration would allow the vaccine to be more easily produced, transported and stored. A dried vaccine promises to reduce the cost of the vaccine which is an important economic factor when producing millions of vaccine doses for at risk populations. Additionally, a dried vaccine may accelerate a concerted world-wide effort to eradicate this devastating disease.