Cryopreservation of Anopheles Embryos

描述(由申请人提供)：理想的、最有效的消除恶性疟原虫(PF)的工具是一种高效的疫苗，它可以防止血液阶段感染，从而防止疾病和传播。恶性疟原虫是99%的疟疾死亡病例的病原体。Sanaria的目标是开发一种PF子孢子(SPZ)疫苗并将其商业化，该疫苗可以防止90%的接受者发生PF血液期感染。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还促进了PfSPZ的制造，以用于挑战感染以测试疫苗和药物(PfSPZ挑战)和用于化学预防下的挑战接种(PfSPZ-CVac)。这些产品组合正在积极地商业化。所有这些PfSPZ产品的生产都依赖于斯氏按蚊，这些蚊子可以从卵到成虫的无菌饲养存活下来，并在它们的唾液腺中产生大量的PfSPZ(&gt；105)。Sanaria的生产过程中一个脆弱的方面是它依赖只能在群体中维持的蚊子；这样的群体容易崩溃，随着时间的推移易受基因漂移的影响，从而面临永久失去所需基因和表型的风险。这是因为尽管经过了几十年的努力，但在我们取得第一阶段的突破性成果之前，活着的按蚊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，即冷冻保存斯氏艾美耳球虫(SDA500-9800株)卵子的能力。在GMP的背景下，能够超低温保存大量的卵子将允许Sanaria通过使用主蚊子库开始受控生产，从而使我们能够重复地返回由具有所需遗传背景的相同定义的、具有特征的材料产生的储存库。如果精心管理，这样的库存可以持续几十年。在此第二阶段项目中，我们将全面优化冷冻保存方法(特定目标1)，并将该方法用于扩大生产，最初是为了确保用于制造目的的菌落的安全性(特定目标2)，随后是为了生产更大的主蚊子卵库(特定目标3)，所有用于制造的蚊子都将来自该库。特色化的主蚊子卵库的详细信息将作为生物制品主文件提交给FDA。我们还将系统地应用卵子冷冻技术，以支持斯氏疟原虫改良品系的发展，并在培育出此类品系的卵子后建立卵库(具体目标4)。该项目的成功将对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.