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

PfSPZ 疫苗的模块化生产：在蚊子和体外生产 PfSPZ 的动合生产

• 批准号: 10761373
• 负责人: STEPHEN Lev HOFFMAN
• 金额: $ 28.22万
• 依托单位: SANARIA, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-06 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10761373
• 关键词: Address; African; Anopheles Genus; Antigens; Biological; Biological Assay; Bioreactors; Blood; Chitinase; Complex; Country; Cryopreservation; Culicidae; Dissociation; Divorce; Excision; Female; Fluorescent Dyes; Future; Germ Cells; Goals; Good Manufacturing Process; Grant; Human; In Vitro; Infection; Link; Location; Magnetism; Malaria; Malaria Vaccines; Membrane; Membrane Proteins; Methods; Nature; Oocysts; Parasites; Parasitic infection; Plasmodium falciparum; Plasmodium falciparum vaccine; Pregnant Women; Process; Production; Protocols documentation; Quality Control; Readiness; Serum; Specific qualifier value; Sporozoite vaccine; Sporozoites; Standardization; Structure; Survival Rate; Testing; Time; Vaccination; Vaccine Production; Vaccines; Work; cell motility; comparative; cost; density; extracellular; feeding; flexibility; large scale production; malaria infection; malaria transmission; male; manufacture; matrigel; prevent; timeline; transmission process; uptake; vaccine efficacy; zygote

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.

2022年，世卫组织呼吁开发预防疟原虫的高效疟疾疫苗 恶性疟原虫（Pf）感染> 90%。在疟疾疫苗中，Sanaria® PfSPZ（Pf 子孢子（SPZ））疫苗已经显示出针对对照人类> 90%的疫苗效力（VE 疟疾感染和维生素E对孕妇的疟疾密集现场传播， 传输多年，没有提升。我们的目标是进一步提高效力、提高效率 生产一致性和降低商品成本（COGS），以便能够使用PfSPZ疫苗 来消灭疟疾。PfSPZ疫苗使用阶段V Pf配子母细胞生产， 蚊子，一个过程，包括密切协调培养配子体和蚊子 养育最重要的是，Sanaria已经开发出生产PfSPZ的方法， 在生物反应器中（Nature，2022）。过渡到生物反应器生产将提高生产效率， 生产至少10倍，减少COGS> 90%，并允许PfSPZ疫苗生产 国际吧本提案旨在提供大量生产的、无菌的、纯化的、冷冻保存的 可用于提高蚊子和生物反应器产生的 PfSPZ疫苗。我们将优化生产、纯化、冷冻保存、QC放行和在 动合子的制造。制造业中以动合子结束的早期步骤可以被取消， 从动合子开始的后续步骤联系起来，允许两者单独进行， 不受时间或物理位置的限制。工作将在四个具体目标中进行。一曰： 优化动合子生产。将优化V期配子母细胞的培养基和培养条件 转化为动合子读数将包括雌性：雄性比例、巨配子体密度和 阶段特异性抗原表达。我们的目标是一个方法，始终导致25%的转化率 从第五阶段的配子母细胞到动合子。2.发展和优化动合子纯化方法。我们 将测试多种方法，选择那些可以扩展的方法。我们的目标是Pf动合子 100%不含红细胞，使用可适应大规模生产的方法，产率为80% 制造业3：制定动合子冷冻保存方案。Sanaria常规冷冻保存 PfSPZ具有一致的、出色的结果。动合子与PfSPZ相似。两者都是能动的， 多膜薄膜复合体，表达一种占优势的膜蛋白，大小相似。一 冷冻保护剂添加剂（CPA）、冷却和升温速率以及CPA稀释方法的范围将在 测试.我们的目标是至少50%的存活率。4：开发动合子活力测定法， 力量将测试4种方法：a）膜完整性，B）运动性，c）对蚊子的免疫力， 产生PfSPZ，和d）动卵细胞体外转化为3天和8天卵囊和iPfSPZ。