Cryopreservation of Anopheles stephensi embryos

描述(申请人提供)：疟疾每年造成数亿病例和100多万人死亡。一种高效的疫苗可以预防血液期感染，从而防止所有疾病和传播，这将是消除恶性疟原虫(PF)的理想工具，恶性疟原虫是导致99%疟疾死亡的原因。通过受感染蚊子的叮咬给予减毒肺泡子孢子虫(PfSPZ)，可使90%的人类志愿者免受持续至少10个月的实验性肺孢子虫攻击。Sanaria的目标是开发一种减毒PfSPZ疫苗并将其商业化，该疫苗可以防止90%的接受者发生肺泡炎血液期感染--具有这些特征的疫苗可以用于从世界上消除肺炎病。这种疫苗有可能带来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临床试验的批准(IND)，并于2009年5月启动了一期临床试验，以评估PfSPZ疫苗的安全性、免疫原性和保护效果。计划在非洲成年人、幼儿和婴儿中进行更多的概念验证研究，随后将进行第二阶段研究，然后是关键的第三阶段研究，以支持许可。Sanaria的制造过程使用冷冻保存的感染恶性疟原虫的红细胞的主细胞库作为寄生虫的来源，并使用斯氏按蚊来饲养PfSPZ。然而，由于没有方法冷冻保存按蚊生命周期的任何阶段，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。将使Sanaria能够冷藏不同菌株的An。Stphensi，降低与菌株维护相关的成本，生产统一的种子库，并提供防止菌株损失或遗传漂移的安全。转基因抗癌基因的冷藏库。Stephensi和其他按蚊。还将为那些旨在使用转基因或不育蚊子控制传播的人提供直接好处。在试点研究中，我们已经表明。使用一种新型的冷冻保护剂(CPA)混合物，可以在液氮的气相中超低温保存斯氏沼虾，解冻后成功孵化和幼体发育。一项描述导致生产第一个成功的按蚊卵子冷冻保存的细节的发明公开已经提交。到目前为止，与同一批卵子的非冷冻对照相比，孵化率为10%，这清楚地表明了概念的验证。这一建议将定义和优化成功的超低温保存。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。成功的结局将以冷冻保存的AN卵子的发育情况来衡量。对唾液腺中的PfSPZ感染保持完全敏感性的斯氏杆菌。An的其他菌株。Sanaria持有的Stephensi，包括转基因菌株，将被类似地超低温保存。 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。Sanaria的目标是为三个主要市场开发和商业化90%保护性疟疾疫苗，年收入可能达到10亿美元。该项目的成功将代表着媒介和疟疾生物学领域的重大突破，将确保保护Sanaria在疫苗生产过程中使用的蚊子的遗传存量的能力，将推动Sanaria朝着确保不同生产地点蚊子群体的一致性的方向发展，并将提供一个安全的冷藏库，从该材料可以定期启动蚊子生产，并可以在明确的稳定性计划中遵循该材料。

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.