Enhancement of gametocytogenesis in Plasmodium falciparum by genetic engineering for improved PfSPZ Vaccine Manufacture

世卫组织估计，2018年，疟疾在全球造成约2.28亿例临床发作和约40.5万例死亡。 尽管每年投资超过30亿美元用于控制措施，但2018年是连续第三年， 疟疾病例没有减少，表明实施进一步影响的能力已经饱和， 现有的战略。对高效疟疾疫苗的迫切医疗需求尚未得到满足 防止感染和疾病。Sanaria的第一代疫苗基于辐射减毒，无菌， 纯化、冷冻保存的恶性疟原虫（Pf）子孢子（SPZ）PfSPZ疫苗已在 在非洲6个国家、欧洲2个国家和美国进行了19项临床试验，并获得了快速通道 FDA的认定。符合3期标准的疫苗的临床试验将于2020年年中开始， 计划于2022年在美国（FDA）和欧洲（EMA）获得上市许可。PfSPZ疫苗是靶向的 在非洲的旅行者和居民中预防疟疾，并为整个社区接种疫苗， 在非洲的重点地区消灭疟疾。在未来5-10年，我们 旨在显著提高PfSPZ疫苗的效力并降低其商品成本（COG）， 可以最佳地用于预防Pf疟疾。我们的战略之一是提高我们 疫苗将包括来自地理上不同地区的其他Pf菌株，如Pf 7 G8（巴西）和 PfNF135.C10（柬埔寨）。除PfNF 54（西非毒株）外，在人类中评估的所有其他Pf毒株均为 可怜的配子体生产者。提高大规模生产PfSPZ效率的途径 不同的地理区域和减少的COG将产生更多的肥沃的土壤， 每单位培养物中的配子体用于在蚊子中产生PfSPZ。在自然界中， 仅在一小部分血液期寄生虫中，由于配子体发生相关的表观遗传抑制， 基因.控制配子体的承诺将提供一个强大的工具，提高生产PfSPZ。一 这个过程中的关键分子是主开关转录因子PfAP 2-G，其表达 与给定Pf菌株产生的配子母细胞的百分比直接相关。该基因的缺失 完全消除配子体的产生。我们建议增加配子体的生产能力， Sanaria的疫苗株，并通过使用过量表达该基因来减少PfSPZ生产COG。 CRISPR-Cas9基因编辑以修饰pfap 2-g的3 '-UTR中的配对元件（PE）。作为替代方案，我们 也将通过用组成型钙调蛋白替换PfAP 2-G的启动子来减轻PfAP 2-G的表观遗传沉默 启动子在条件调控配子体诱导（开/关）系统，我们已经开发。我们将 产生来自不同地理区域的Pf的增强的配子体产生系，比较和评估 具有增强的配子体发生的克隆，并选择那些具有最佳体外生长的克隆， 无菌蚊中感染性PfSPZ的患病率和强度。

The WHO estimates that in 2018, malaria caused ~228M clinical episodes and ~405,000 deaths worldwide. Despite an annual investment of >$3 billion for control measures, 2018 was the 3rd consecutive year in which there was no decrease in malaria cases, indicating a saturation of capacity to implement further impact with currently available strategies. There is an urgent unmet medical need for a highly efficacious malaria vaccine that prevents infection and disease. Sanaria’s 1st generation vaccine based on radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum (Pf) sporozoites (SPZ) PfSPZ Vaccine has been assessed in 19 clinical trials in 6 countries in Africa, 2 countries in Europe, and the US, and received Fast Track designation from the FDA. Clinical trials with Phase 3 compliant vaccine will begin in mid 2020, and licensure (marketing authorization) in the US (FDA) and in Europe (EMA) is planned for 2022. PfSPZ Vaccine is targeted to prevent malaria in travelers to and residents of Africa, and for immunizing the entire community to halt transmission and eliminate malaria from geographically focused areas of Africa. During the next 5-10 years, we aim to significantly increase potency and decrease cost of goods (COGs) of PfSPZ-based vaccines so they can be optimally used to prevent Pf malaria. One of our strategies to improve the breadth of protection in our vaccines is to include additional strains of Pf from geographically diverse regions such as Pf7G8 (Brazil) and PfNF135.C10 (Cambodia). Other than PfNF54 (West Africa strain), all other Pf strains assessed in humans are poor gametocyte producers. An approach to improving the efficiency of large-scale production of PfSPZs from different geographic regions and decreasing COGs would be generating increased numbers of fertile gametocytes per unit of culture for production of PfSPZ in mosquitoes. In nature, gametocytogenesis occurs only in a small subset of blood stage parasites due to epigenetic suppression of gametocytogenesis-related genes. Control of gametocyte commitment would provide a powerful tool for improving production of PfSPZ. A key molecule in this process is the master switch transcription factor, PfAP2-G, the expression of which correlates directly with the percent of gametocytes produced by a given Pf strain. Deletion of this gene completely abolishes gametocyte production. We propose to increase the gametocyte production capacity of Sanaria’s vaccine strains and decrease PfSPZ manufacturing COGs by over-expressing this gene by using CRISPR-Cas9 gene editing to modify the pairing elements (PE) in the 3’-UTR of pfap2-g. As an alternative, we will also relieve epigenetic silencing of PfAP2-G by replacing its promoter with the constitutive calmodulin promoter in a conditionally regulatable gametocyte induction (on/off) system we have developed. We will generate enhanced gametocyte-producing lines of Pf from different geographic regions, compare and evaluate clones with enhanced gametocytogenesis and select those with optimal in vitro growth that maintain high prevalence and intensities of infectious PfSPZs in aseptic mosquitoes.