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， 第一种对抗人类寄生虫病的疫苗PfSPZ疫苗由无菌、减毒、 代谢活性的、纯化的、小瓶化的和冷冻保存的恶性疟原虫（Pf）子孢子（SPZ）。 PfSPZ疫苗的生产与PfSPZ攻毒的所有步骤（除辐射衰减外）相同， 被世界各地的研究人员用于控制人类疟疾感染，以评估实验性疟疾药物， 候选疫苗和对疟疾的天然免疫或抗性。第三种临床试验产品是 PfSPZ-CVac，PfSPZ激发在化学预防性覆盖下给药。PfSPZ疫苗可预防 恶性疟原虫（Pf）同源（同株）和异源（异株）疟疾临床研究 在美国进行的试验，德国坦桑尼亚和马里。到目前为止，80-100%的保护持续至少8 已证实对异源性攻击感染的有效期为3个月，对同源性攻击感染的有效期为14个月。 正在加蓬、马里和赤道几内亚进行的试验正在测试优化的方案，为3期试验做准备。 2020年开始的临床试验，以支持2021年向FDA提交的生物制品许可申请（BLA）。所有PfSPZ 产品是在无菌饲养的蚊子中生产的，这些蚊子的卵是从 持续维持的斯氏按蚊群体。该项目的目标是提交一份生物制品 Sanaria冻存A的主文件。stephensi SDA 500菌株，以支持PfSPZ的生产。 并使用来自冷冻保存的蚊子生产一批PfSPZ攻毒 卵，并适应大规模冷冻保存的A.冈比亚。这其中的根本 这一过程是在完全符合cGMP的情况下从冷冻保存的卵库中提高蚊子产量。 这项工作将包括以巨大的规模冷冻保存卵子和产生一个主卵子 MEB和工作卵库（WEB）。stephensi SDA 500鸡蛋。这项工作是 纳入三个具体目标：1）完成符合GMP的MEB和WEB生产， 将需要开发新的放行和稳定性试验，生成SOP和检验报告， 建立冻存卵子的稳定性计划，并向 FDA; 2）使用来自冷冻保存WEB的蚊子生产一批PfSPZ攻毒， 用于临床试验;和3）冷冻保存A。冈比亚大规模产卵以支持蚊子释放计划。 按蚊卵的冷冻保存是一项突破性的使能技术， 按蚊研究，特别是在需要保留转基因菌株的实验室， 但资源有限，只能这样做。

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.