Modularizing manufacture of PfSPZ vaccines: ookinete production for PfSPZ manufacture in mosquitoes and in vitro

2022年，世卫组织呼吁开发预防疟疾的高效疫苗 90%的受者体内存在恶性疟原虫感染。在疟疾疫苗中独一无二的是Sanaria®PfSPZ(PF 子孢子体(SPZ)疫苗对受控人类显示出90%的疫苗效力(VE) 疟疾感染和VE预防现场疟疾向孕妇的两次强烈传播 变速箱几年没有增压。我们的目标是进一步增加效力，提高效率 以及生产的一致性和降低商品成本(COGS)以能够使用PfSPZ疫苗 用于消灭疟疾。PfSPZ疫苗是使用V期PF配子体喂养到 蚊子，这一过程包括配子细胞培养和蚊子之间的紧密协调 养育。最重要的是，Sanaria已经开发出不含蚊子的PfSPZ生产方法 在生物反应器中(自然，2022)。过渡到生物反应器生产将提高 生产至少减少10倍，COGS减少90%，并允许生产PfSPZ疫苗 全世界。本提案旨在提供大规模生产、无菌、提纯、低温保存 可用于提高蚊子和生物反应器产生的生产效率的动植物 PfSPZ疫苗。我们将优化生产、提纯、冷冻保存、QC释放和使用 动植物的制造。以动植物为终点的制造的早期步骤可以被取消- 与以卵母细胞开始的后续步骤相联系，允许两者单独进行， 不受时间或物理位置的限制。这项工作将分四个具体目标进行。1： 优化动植物生产。V期配子体的培养液和培养条件将得到优化 用来转化为动植物。读数将包括雌雄比、大配子密度和 阶段特异性抗原表达。我们的目标是一个始终如一地实现25%转化率的方法 从V期配子体到卵动子。2.开发和优化动细胞纯化方法。我们 将测试多种方法，选择可扩展的方法。我们的目标是PF动植物 100%不含红细胞，产率为80%，方法适用于大规模 制造业。3：制定卵母细胞冷冻保存方案。萨纳里亚常规的冷冻保存 PfSPZ具有一致的、出色的结果。动植物与PfSPZ相似。两者都是能动的，有一个 多膜复合体，表达主要的膜蛋白，大小相似。一个 冷冻保护剂添加剂(CPA)的范围、冷却和升温速度以及CPA稀释方法将包括 测试过。我们的目标是存活率至少达到50%。4：开发动细胞活力的检测方法和 威力。将测试4种方法：a)膜完整性，b)运动性，c)对蚊子的感染性 产生PfSPZ，以及d)体外将卵母细胞转化为3天和8天的卵囊和IPfSPZ。

In 2022 WHO called for highly efficacious vaccines against malaria that prevent Plasmodium falciparum (Pf) infection in > 90% of recipients. Alone among malaria vaccines, Sanaria® PfSPZ (Pf sporozoite (SPZ)) vaccines have shown > 90% vaccine efficacy (VE) against controlled human malaria infection and VE against intense field transmission of malaria to pregnant women for two transmission years without boosting. Our goals are to further increase potency, increase efficiency and consistency of manufacture and reduce cost of goods (COGS) to be able to use a PfSPZ vaccine for elimination of malaria. PfSPZ vaccines are manufactured using stage V Pf gametocytes fed to mosquitoes, a process that includes tight coordination between culture of gametocytes and mosquito rearing. Most importantly, Sanaria has developed methods for producing PfSPZ without mosquitoes in bioreactors (Nature, 2022). Transitioning to bioreactor production would increase efficiency of manufacture by at least 10-fold, reduce COGS by >90%, and allow for PfSPZ vaccine production worldwide. This proposal is intended to provide mass-produced, aseptic, purified, cryopreserved ookinetes that can be used to increase efficiency of production of mosquito- and bioreactor-produced PfSPZ vaccines. We will optimize production, purification, cryopreservation, QC release, and use in manufacturing of ookinetes. The early steps in manufacturing that end with ookinetes can then be de- linked from the later steps that commence with ookinetes, allowing both to proceed separately, unconstrained by timing or physical location. The work will be addressed in 4 Specific Aims. 1: Optimize ookinete production. Media and culture conditions for stage V gametocytes will be optimized for conversion to ookinetes. Readouts will include female:male ratio, macrogametocyte density, and stage specific antigen expression. Our target is a method that consistently results in 25% conversion of stage V gametocytes to ookinetes. 2: Develop and optimize methods for ookinete purification. We will test multiple methods, selecting those that can be scaled. Our target is Pf ookinetes that are 100% free of RBCs with a yield of 80% using a method that can be adapted to large scale manufacturing. 3: Develop a protocol for ookinete cryopreservation. Sanaria routinely cryopreserves PfSPZ with consistent, excellent results. Ookinetes are similar to PfSPZ. Both are motile, have a multimembrane pellicular complex, express a dominant membrane protein, and are similar in size. A range of cryoprotectant additives (CPA), cooling and warming rates, and CPA dilution methods will be tested. Our target is a survival rate of at least 50%. 4: Develop assays for ookinete viability and potency. 4 approaches will be tested: a) Membrane integrity, b) Motility, c) Infectivity to mosquitoes to produce PfSPZ, and d) In vitro conversion of ookinetes to 3- and 8-day oocysts and iPfSPZ.