Cryopreservation of Anopheles stephensi embryos

史氏按蚊胚胎的冷冻保存

• 批准号: 8117546
• 负责人: Peter F. Billingsley
• 金额: $ 30万
• 依托单位: SANARIA, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8117546
• 关键词: Adult; Africa; African; Anopheles Genus; Attenuated; Biology; Biotechnology; Bite; Blood; Cessation of life; Characteristics; Child; Clinical; Clinical Trials; Cryopreservation; Culicidae; Development; Disclosure; Embryo; Ensure; Erythrocytes; Fertility; Genetic; Genetic Drift; Goals; Health; Human Volunteers; Infant; Infection; Institutes; Lead; Licensure; Life Cycle Stages; Liquid substance; Longevity; Maintenance; Malaria; Malaria Vaccines; Marketing; Maryland; Measures; Methods; Military Personnel; Nitrogen; Outcome; Parasites; Phase; Phase I Clinical Trials; Pilot Projects; Plasmodium falciparum; Predisposition; Preparation; Procedures; Process; Production; Protocols documentation; Quality Control; Safety; Salivary Glands; Secure; Security; Seeds; Site; Small Business Innovation Research Grant; Source; Sporozoite vaccine; Sporozoites; Staging; Sterility; Technology; Testing; Transgenic Organisms; Universities; Vaccines; cell bank; clinical lot; cost; disease transmission; egg; hatching; immunogenicity; manufacturing process; mortality; novel; phase 2 study; phase 3 study; prevent; programs; protective efficacy; scale up; success; tool; transmission process; vapor; vector; young adult

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.

描述（由申请人提供）：疟疾每年导致数亿病例和超过 100 万人死亡。一种高效的疫苗可以预防血期感染，从而预防所有疾病和传播，这将是消除恶性疟原虫 (Pf) 的理想工具，而恶性疟原虫是 99% 疟疾死亡的原因。通过受感染的蚊子叮咬给予减毒 Pf 子孢子 (PfSPZ)，可以保护超过 90% 的人类志愿者免受实验性 Pf 攻击，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，该疫苗可预防超过 90% 的受者出现 Pf 血期感染 - 具有这些特性的疫苗可用于消除世界上的 Pf。这种疫苗的年收入有可能超过 10 亿美元。 Sanaria 已成功建立稳健、可重复且一致的 PfSPZ 疫苗临床批次生产和发布，获得 FDA 临床试验批准 (IND)，并启动 1 期临床试验以评估 PfSPZ 的安全性、免疫原性和保护功效疫苗于 2009 年 5 月上市。计划在非洲成人、幼儿和婴儿中进行额外的概念验证研究，随后将进行第 2 阶段研究，然后是关键的第 3 阶段研究，以支持许可。 Sanaria 的制造过程利用冷冻保存的感染恶性疟原虫的红细胞主细胞库作为寄生虫来源，并利用斯蒂芬按蚊来培养 PfSPZ。然而，由于没有方法可以冷冻保存按蚊生命周期的任何阶段，因此用于生产 PfSPZ 疫苗的蚊子来自持续维持的蚊群。按蚊卵的冷冻保存。将使 Sanaria 能够冷冻储存不同的 An 菌株。斯蒂芬西，降低与菌株维护相关的成本，生产统一的种子库存并提供防止菌株损失或遗传漂变的保障。转基因 An 的冷冻银行。史蒂芬西和其他按蚊属。还将为那些打算使用转基因或不育蚊子来控制传播的人提供直接利益。在试点研究中，我们已经表明 An。可以使用新型冷冻保护添加剂 (CPA) 混合物在液氮气相中冷冻保存史蒂芬西，并在解冻后成功孵化和幼虫发育。一项发明公开描述了第一个成功生产按蚊卵冷冻保存的细节，该发明已提交。与同一批次鸡蛋的非冷冻保存对照相比，迄今为止的孵化率为 10%，这清楚地表明了概念验证。该提案将定义和优化 An 的成功冷冻保存。斯蒂芬西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。成功的结果将通过冷冻 An 卵的发育来衡量。 Stephensi 保留对唾液腺中 PfSPZ 感染的完全易感性。 An 的其他菌株。 Sanaria 持有的史蒂芬西病毒，包括转基因菌株，也将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 1-300 万人死亡，每年造成非洲 GDP 损失超过 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并商业化保护性超过 90% 的疟疾疫苗，年收入潜力超过 10 亿美元。该项目的成功将代表媒介和疟疾生物学领域的重大突破，将确保能够保存 Sanaria 在疫苗生产过程中使用的蚊子的遗传资源，并将推动 Sanaria 确保不同地区蚊子群体的一致性。生产基地，并将提供安全的材料冷冻库，可以定期启动蚊子生产，并可以遵循明确的稳定性计划。