Cryopreservation of Anopheles Embryos

摘要：PfSPZ疫苗目前有望成为世界上第一个获得许可的疟疾疫苗和 第一种针对人类寄生虫病的疫苗。PfSPZ疫苗由无菌、减毒、 代谢活跃、纯化、瓶装和冷冻保存的恶性疟原虫(PF)子孢子(SPZ)。 PfSPZ疫苗的制造与PfSPZ挑战共享所有步骤(辐射衰减除外)，即 被世界各地的研究人员用来控制人类疟疾感染来评估实验性的疟疾药物， 候选疫苗，以及对疟疾的自然免疫力或抵抗力。第三种正在进行临床测试的产品是 PfSPZ-CVAc，PfSPZ挑战在化学预防的覆盖下进行。PfSPZ疫苗已经预防了 恶性疟的同源(同株)和异源(异株)临床研究 试验在美国、德国、坦桑尼亚和马里进行。到目前为止，80%-100%的保护持续至少8 已经证明，对抗异源挑战感染数月，对抗同源挑战感染14个月。 在加蓬、马里和赤道几内亚正在进行的试验正在测试优化的方案，为第三阶段做准备 临床试验将于2020年开始，以支持2021年向FDA提交的生物制品许可证申请(BLA)。所有PfSPZ 产品是在无菌条件下饲养的蚊子中生产的，这些蚊子是从一个 持续维持的斯氏按蚊群体。该项目的目标是提交一个生物制品 Sanaria的冷冻保存的S.stephensi SDA500菌株的主文件，以支持PfSPZ的制造- 用超低温保存的蚊子制作一批PfSPZ挑战赛 并将该方法应用于冈比亚按蚊的大规模超低温保存。这是最基本的 这一进程是在完全符合cGMP的情况下，提高冷冻卵库的蚊子产量。 这项工作将涉及极大规模的卵子冷冻保存和母卵的产生。 建立了斯氏拟青霉SDA500卵子超低温保存库(MEB)和工作卵库(Web)。这项工作是 纳入三个具体目标：1)完成符合GMP的MEB和WEB的制造， 将需要开发新的释放和稳定性分析，生成标准操作规程和分析证书， 制定冷冻卵子的稳定性计划，并向美国食品和药物管理局提交生物制品主文件 FDA；2)使用从冷冻保存的网络中提取的蚊子，制造一批PfSPZ挑战赛 用于临床试验；以及3)大规模冷冻冈比亚按蚊卵子，以支持蚊子释放计划。 冷冻保存按蚊卵子是一项突破性的使能技术，将可用于支持 蚊子研究，特别是在需要保留转基因菌株和 线路，但这样做的资源有限。

ABSTRACT: PfSPZ Vaccine is currently on track to be the world’s first licensed vaccine against malaria and the first vaccine against a human parasitic disease. PfSPZ Vaccine is composed of aseptic, attenuated, metabolically active, purified, vialed and cryopreserved Plasmodium falciparum (Pf) sporozoites (SPZ). Manufacture of PfSPZ Vaccine shares all steps (except radiation attenuation) with PfSPZ Challenge that is used by researchers worldwide in controlled human malaria infections to evaluate experimental malaria drugs, candidate vaccines, and natural immunity or resistance to malaria. The third product under clinical testing is PfSPZ-CVac, PfSPZ Challenge given under chemoprophylactic cover. PfSPZ Vaccine has protected against homologous (same strain) and heterologous (different strains) of Plasmodium falciparum (Pf) malaria in clinical trials conducted in the U.S., Germany, Tanzania, and Mali. To date, 80–100% protection lasting for at least 8 months against heterologous and 14 months against homologous challenge infections has been demonstrated. Ongoing trials in Gabon, Mali, and Equatorial Guinea are testing optimized regimens in preparation for Phase 3 clinical trials starting in 2020 in support of a Biologics License Application (BLA) to the FDA in 2021. All PfSPZ products are produced in mosquitoes reared aseptically from disinfected eggs that are harvested from a continuously-maintained Anopheles stephensi colony. The goals of this project are to submit a Biologics Master File for Sanaria’s cryopreserved A. stephensi SDA500 strain in support of the manufacture of PfSPZ- based products and to manufacture one lot of PfSPZ Challenge using mosquitoes derived from cryopreserved eggs, and also to adapt the methodology for large scale cryopreservation of A. gambiae. Fundamental to this process is the elevation of mosquito production from banks of cryopreserved eggs at full cGMP compliance. The work will involve cryopreservation of eggs at hugely increased scale and the generation of a master egg bank (MEB) and working egg bank (WEB) of cryopreserved A. stephensi SDA500 eggs. The work is incorporated into three specific aims: 1) Complete the GMP-compliant manufacture of a MEB and WEB which will entail development of novel release and stability assays, generation of SOPs and Certificates of Analysis, establishment of a stability program for cryopreserved eggs and submission of a Biologics Master File to the FDA; 2) Manufacture, using mosquitoes derived from the cryopreserved WEB, one lot of PfSPZ Challenge for use in clinical trials; and 3) Cryopreserve A. gambiae eggs at scale to support mosquito release programs. Cryopreservation of Anopheles eggs is a breakthrough enabling technology that will be available to support Anopheles mosquito research, particularly in laboratories that need to retain genetically modified strains and lines but have limited resources to do so.