In vitro bioreactor production of a genetically modified late liver stage-arresting replication competent Plasmodium falciparum sporozoite vaccine

摘要 2020年，疟疾造成2.41亿例临床病例和62.7万例死亡，比2019年显著增加。那里 在非洲死于疟疾的人数超过了死于COVID-19的人数。有一个迫切的未满足的医疗需求， 预防个人感染和疾病的疟疾疫苗，可用于大规模接种 消灭疟疾的计划。根据在50多万名婴儿中开展的试点实施方案的结果， RTS，S/AS 01于2021年底获世卫组织推荐用于5个月大婴儿的免疫接种，共接种4次 在21个月内进行管理。RTS，S显著减少了21%的疟疾住院率， 疟疾减少30%它没有显著降低脑型疟疾、严重疟疾贫血或总体死亡率，或 预防恶性疟原虫（Pf）感染。在正在开发的疫苗中，只有Sanaria的PfSPZ疫苗 具有抗Pf感染的功效，可考虑用于预防个体中的Pf感染或用于 以地理为重点的消灭疟疾运动。Sanaria的第一代疫苗PfSPZ疫苗是 由放射减毒的恶性疟原虫（Pf）子孢子（SPZ）组成，其在肝脏中早期停止 阶段3期临床试验将于2022年年中开始，并在欧洲（EMA）和美国获得上市许可 (FDA)计划于2023/2024年。Sanaria的第二代疫苗是PfSPZ-CVac（化学预防 疫苗）。在PfSPZ-CVac中，寄生虫在肝脏中复制，生物学上放大免疫原负荷， 到50,000倍，然后被抗疟疾药物杀死。PfSPZ-CVac与氯喹联合给药 (CQ)，对异源受控人疟疾感染（CHMI）产生100%的疫苗效力（VE）12 在使用22%剂量PfSPZ接种后10周，需要达到80%的抗异源CHMI VE PfSPZ疫苗PfSPZ-CVac（CQ）在约1/5剂量下比PfSPZ疫苗更具保护性。然而，在这方面， 在PfSPZ第一次给药后可能出现短暂的疟疾症状，如果CQ给药不当， 血液中的寄生虫繁殖可能导致严重的疟疾。为了保持PfSPZ的增强的效力， CVac和消除其缺点，我们遗传改变Pf能够完全复制，但逮捕之前， 通过删除一个基因进入血液我们的第一个后期逮捕复制能力（LARC） PfSPZ-LARC 1在具有人源化肝脏的小鼠和人类中完全减毒。到 为了增加PfSPZ-LARC疫苗的潜在安全性，我们创造了一种缺失两个基因的寄生虫，PfSPZ- LARC 2，也在肝脏阶段晚期停止。Sanaria还实现了PfSPZ的体外生产， 称为iPfSPZ，从而消除了在生产PfSPZ疫苗中对蚊子的需要， 显著提高效率并降低生产PfSPZ疫苗的成本。在这个项目中，我们将 优化iPfSPZ-LARC 2的体外生产和纯化，生产无菌、纯化、冷冻保存的iPfSPZ- LARC 2，并证明这些iPfSPZ-LARC 2是感染性的，并可以发展到晚期肝脏阶段，但 减弱的在下一阶段，我们将生产iPfSPZ-LARC 2用于临床试验评估。

ABSTRACT In 2020 malaria caused 241M clinical episodes and 627,000 deaths, a significant increase from 2019. There were more deaths in Africa from malaria than from COVID-19. There is an urgent unmet medical need for a malaria vaccine that prevents infection and disease in individuals and can be deployed in mass vaccination programs for malaria elimination. Based on results of a pilot implementation program in >500,000 infants, RTS,S/AS01 was recommended in late 2021 by WHO for immunization of 5-month-olds with 4 immunizations administered over 21 months. RTS,S significantly reduced hospital admissions for malaria by 21% and severe malaria by 30%. It did not significantly reduce cerebral malaria, severe malaria anemia, or overall mortality, or prevent Plasmodium falciparum (Pf) infection. Of vaccines under development, only Sanaria’s PfSPZ vaccines have the efficacy against Pf infection to be considered for prevention of Pf infection in individuals or for geographically focused Pf malaria elimination campaigns. Sanaria’s 1st generation vaccine, PfSPZ Vaccine, is comprised of radiation-attenuated Plasmodium falciparum (Pf) sporozoites (SPZ), which arrest early in the liver stage. Phase 3 clinical trials will begin in mid 2022, and marketing authorizations in Europe (EMA) and the US (FDA) are planned for 2023/2024. Sanaria’s 2nd generation vaccine is PfSPZ-CVac (Chemoprophylaxis Vaccine). In PfSPZ-CVac, the parasites replicate in the liver, biologically amplifying the immunogen load by up to 50,000-fold and then are killed by an anti-malarial drug. PfSPZ-CVac co-administered with chloroquine (CQ), gave 100% vaccine efficacy (VE) against heterologous controlled human malaria infection (CHMI) 12 weeks after vaccination using 22% the dose of PfSPZ needed to achieve 80% VE against heterologous CHMI with PfSPZ Vaccine. PfSPZ-CVac (CQ) is more protective than PfSPZ Vaccine at ~1/5 the dose. However, transient symptoms of malaria can occur after 1st dose of PfSPZ, and if CQ is not administered appropriately, parasite multiplication in the blood could cause severe malaria. To retain the enhanced potency of PfSPZ- CVac and eliminate its drawbacks, we genetically altered Pf to be able to fully replicate, but arrest prior to entering the blood by deleting a single gene. The first of our late arresting replication competent (LARC) parasites called PfSPZ-LARC1, was completely attenuated in mice with humanized livers and in humans. To increase the potential safety of PfSPZ-LARC vaccines, we created a parasite with two genes deleted, PfSPZ- LARC2, which also arrests late in the liver stage. Sanaria has also achieved in vitro production of PfSPZ, referred to as iPfSPZ, thereby eliminating the need for mosquitoes in manufacturing PfSPZ vaccines and significantly increasing efficiency and decreasing costs of producing PfSPZ vaccines. In this project we will optimize in vitro production and purification of iPfSPZ-LARC2, produce aseptic, purified, cryopreserved iPfSPZ- LARC2 and demonstrate these iPfSPZ-LARC2 are infectious, and can develop into late liver stages but are attenuated. In the next phase, we will manufacture iPfSPZ-LARC2 for assessment in clinical trials.