Cryopreservation of Anopheles Embryos

描述（由申请人提供）：消灭恶性疟原虫（Pf）的理想、最有效的工具，是一种非常有效的疫苗，可防止血液阶段感染，从而预防疾病和传播。恶性疟原虫是导致99%疟疾死亡的病原体。Sanaria的目标是开发并商业化一种Pf孢子子（SPZ）疫苗，该疫苗可在90%的接种者中预防Pf血期感染。Sanaria(R) PfSPZ疫苗由减毒、纯化、无菌、低温保存的PfSPZ组成。为制造疫苗而开发的平台技术也促进了用于攻击感染的PfSPZ的生产，以测试疫苗和药物（PfSPZ challenge）和用于化学预防下的攻击疫苗接种（PfSPZ- cvac）。这些产品组合正处于商业化的积极时间表上。所有这些PfSPZ产品的生产都依赖于能够从卵到成虫无菌饲养并在其唾液腺中产生大量PfSPZ（100 - 105）的斯氏按蚊。萨那利亚制造过程的一个弱点是，它依赖于只能在群体中生存的蚊子；随着时间的推移，这样的菌落很容易崩溃和遗传漂变，因此有永久失去所需基因型和表型的风险。这是因为尽管经过了几十年的努力，直到我们在第一阶段取得突破性成果，活的按蚊仍无法以任何方式保存下来。在第一阶段，我们展示了新的使能技术，即冷冻保存斯蒂芬氏不动杆菌（SDA500-9800菌株）卵子的能力。在GMP背景下，能够冷冻保存大量卵子将使Sanaria能够通过使用Master Mosquito Bank开始控制生产，从而使我们能够反复返回由具有所需遗传背景的相同定义、特征材料产生的储存库库存。如果管理得当，这样的股票可以持续几十年。在这个二期项目中，我们将充分优化冷冻保存方法（Specific Aim 1），并在大规模生产中使用这种方法，最初是为了确保用于生产目的的菌落的安全性（Specific Aim 2），随后是为了生产更大的主蚊卵库（Specific Aim 3），所有用于生产的蚊子都将来自该库。所描述的主蚊卵库的详细信息将作为生物制剂主文件提交给FDA。我们还将系统地应用卵子冷冻保存技术来支持斯蒂芬氏芽孢杆菌改良菌株的开发，并在开发时创建此类菌株的卵子库（具体目标4）。这个项目的成功将对Sanaria的商业成功产生至关重要的影响。

DESCRIPTION (provided by applicant): The ideal, most effective, tool for eliminating Plasmodium falciparum (Pf), the causative agent of 99% of all malaria deaths, is a highly effective vaccine that prevents blood stage infection and thereby prevents both disease and transmission. Sanaria's goal is to develop and commercialize a Pf sporozoite (SPZ) vaccine that prevents Pf blood stage infection in >90% of recipients. The Sanaria(R) PfSPZ Vaccine is composed of attenuated, purified, aseptic, cryopreserved PfSPZ. The platform technology developed to manufacture the vaccine has also facilitated the manufacture of PfSPZ for challenge infections to test vaccines and drugs (PfSPZ Challenge) and for vaccination by challenge under chemoprophylaxis (PfSPZ-CVac). These comprise a portfolio of products that are on an aggressive timeline to commercialization. Manufacture of all these PfSPZ products is dependent on Anopheles stephensi mosquitoes that can survive aseptic rearing from eggs to adults and produce high numbers (>105) of PfSPZ in their salivary glands. A vulnerable aspect of Sanaria's manufacturing process is its reliance on mosquitoes that can only be maintained in colonies; such colonies are susceptible to crashes and to genetic drift over time, thereby being at risk of permanently losing the desired genotype and phenotype. This is because despite decades of effort, until our breakthrough achievement in Phase I, live Anopheles could not be preserved in any way. In Phase I we demonstrated new enabling technology, the capability to cryopreserve eggs of A. stephensi (SDA500-9800 strain). In the GMP context, being able to cryopreserve large lots of eggs will allow Sanaria to inaugurate controlled manufacturing by use of a Master Mosquito Bank, thereby enabling us to return repeatedly to a repository stock generated from the same defined, characterized material with the desired genetic background. With careful management, such a stock can last for many tens of years. In this Phase II project we will fully optimize the cryopreservation method (Specific Aim 1), and use this method in scaled-up production, initially to ensure security of the colony for manufacturing purposes (Specific Aim 2) and subsequently for producing the larger Master Mosquito Egg Bank (Specific Aim 3) from which all mosquitoes used in manufacturing will be sourced. Details of the characterized Master Mosquito Egg Bank will be submitted to the FDA as a Biologics Master File. We will also systematically apply the egg cryopreservation technology to support development of improved strains of A. stephensi and to create banks of eggs of such strains when developed (Specific Aim 4). Success in this project will have a critically important impact on Sanaria's commercial success.