Cryopreservation of Anopheles stephensi embryos

描述（由申请人提供）：疟疾每年造成数亿例病例和100多万人死亡。预防血液阶段感染从而预防所有疾病和传播的高效疫苗将是消除恶性疟原虫（Pf）的理想工具，恶性疟原虫是99%疟疾死亡的原因。通过被感染的蚊子叮咬施用减毒的Pf子孢子（PfSPZ）导致>90%的人类志愿者免受持续至少10个月的实验性Pf攻击。Sanaria的目标是开发和商业化一种减毒PfSPZ疫苗，该疫苗可预防>90%的受者的Pf血液期感染-具有这些特征的疫苗可用于从世界上消除Pf。这种疫苗有可能获得超过10亿美元的年收入。Sanaria已成功建立了PfSPZ疫苗临床批次的稳健、可重复和一致的生产和放行，获得了FDA临床试验批准（IND），并于2009年5月启动了1期临床试验，以评估PfSPZ疫苗的安全性、免疫原性和保护效力。计划在非洲成人、幼儿和婴儿中进行额外的概念验证研究，随后将进行II期研究，然后进行关键的III期研究，以支持许可证。Sanaria的生产工艺利用冷冻保存的恶性疟原虫感染红细胞的主细胞库作为寄生虫来源，并利用斯氏按蚊饲养PfSPZ。然而，由于没有冷冻保存按蚊生命周期任何阶段的方法，用于生产PfSPZ疫苗的蚊子来自持续维持的蚊子群体。按蚊卵的超低温保存。可以让萨纳里亚公司建立不同的安氏杆菌株的冷冻库Stephensi，降低了与菌株保持相关成本，产生了统一的种子库，并提供了防止菌株损失或遗传漂变的安全性。转基因家蚕的冷冻库建立。斯氏按蚊和其它按蚊属（Anopheles spp.）还将为那些旨在使用基因改变或不育蚊子进行传播控制的人提供直接益处。在试点研究中，我们已经表明，一个。使用一种新的冷冻保护剂添加剂（CPA）混合物，可以在液氮的气相中冷冻保存斑蝶，解冻后成功孵化和幼虫发育。描述导致第一个成功的按蚊卵冷冻保存的生产的细节的发明公开已经提交。迄今为止，与来自相同蛋批次的非冷冻保存对照相比，孵化率为10%，这是概念验证的明确指示。该建议将定义和优化成功的冷冻保存An。通过对不同发育阶段的胚胎以及冷冻保存方法和添加剂进行务实和系统的测试，成功的结果将被测量为从冷冻保存的An卵的发育。在唾液腺中保留对PfSPZ感染的完全易感性。其他菌株的一个。将Sanaria持有的stephensi，包括转基因菌株，进行类似的冷冻保存。 公共卫生相关性：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的目标是为三个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。该项目的成功将代表着病媒和疟疾生物学领域的重大突破，将确保能够保护Sanaria在疫苗生产过程中使用的蚊子的遗传资源，将推动Sanaria确保不同生产地点的蚊子种群的一致性，并将提供一个安全的材料冷冻库，可以定期启动蚊子生产，遵循明确的稳定计划。

DESCRIPTION (provided by applicant): Malaria is responsible for several hundred million cases and over one million deaths per year. A highly effective vaccine that prevents blood stage infection and thereby prevents all disease and transmission would be an ideal tool for eliminating Plasmodium falciparum (Pf), the cause of 99% of malaria mortality. Attenuated Pf sporozoites (PfSPZ) administered by the bite of infected mosquitoes, lead to protection of >90% of human volunteers against experimental Pf challenge that 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 with these characteristics could be used to eliminate Pf from the world. This vaccine has the potential for >$1 billion annual revenues. Sanaria has successfully established robust, reproducible, and consistent manufacture and release of clinical lots of its PfSPZ Vaccine, received FDA approval (IND) for clinical trials, and initiated a Phase 1 clinical trial to assess safety, immunogenicity, and protective efficacy of the PfSPZ Vaccine in May 2009. Additional proof of concept studies in African adults, young children, and infants are planned and will be followed by Phase 2 studies, and then pivotal Phase 3 studies to support licensure. Sanaria's manufacturing process utilizes a master cell bank of cryopreserved P. falciparum-infected erythrocytes as a source of parasites, and Anopheles stephensi mosquitoes to raise the PfSPZ. However, because there is no method for cryopreserving any stage of the Anopheles mosquito life cycle, the mosquitoes used for producing the PfSPZ Vaccine come from a mosquito colony that is continuously maintained. Cryopreservation of the eggs of Anopheles spp. would enable Sanaria to cryobank different strains of An. stephensi, reduce costs associated with strain maintenance, produce a uniform seed stock and provide security against strain loss or genetic drift. Cryobanking of transgenic An. stephensi and other Anopheles spp. would also provide direct benefit to those aiming to use genetically altered or sterile mosquitoes for transmission control. In pilot studies, we have shown that An. stephensi can be cryopreserved in the vapor phase of liquid nitrogen using a novel cryoprotectant additive (CPA) mixture, with successful hatching and larval development following thawing. An invention disclosure describing the details that led to the production of the first successful Anopheles egg cryopreservation has been filed. The hatch rate to date has been 10% compared to non-cryopreserved controls from the same egg batch which is clear indication of proof of concept. This proposal will define and optimize the successful cryopreservation of An. stephensi by a pragmatic and systematic testing of embryos at different stages of development and of cryopreservation methods and additives. Successful outcome will be measured as the development from cryopreserved eggs of An. stephensi that retain full susceptibility to infection with PfSPZ in the salivary glands. Other strains of An. stephensi held by Sanaria, including transgenic strains, will be similarly cryopreserved. 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. Success in this project will represent a major breakthrough in the fields of vector and malaria biology, will ensure the ability to conserve the genetic stock of the mosquitoes used by Sanaria's in its vaccine manufacturing process, will move Sanaria towards ensuring consistency of mosquito colonies across different manufacturing sites and will provide a secure cryobank of material from which mosquito production can be initiated periodically and which can be followed in a well-defined stability program.