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

世界卫生组织估计，2018 年，疟疾在全球造成约 2.28 亿例临床病例和约 405,000 人死亡。 尽管控制措施的年度投资超过 30 亿美元，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饱和 目前可用的策略。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弱、无菌、 纯化、冷冻保存的恶性疟原虫 (Pf) 子孢子 (SPZ) PfSPZ 疫苗已在 非洲6国、欧洲2国、美国19项临床试验，获快速通道 FDA 指定。符合第 3 阶段要求的疫苗的临床试验将于 2020 年中期开始，并获得许可 计划于 2022 年在美国 (FDA) 和欧洲 (EMA) 获得（营销授权）。PfSPZ 疫苗的目标是 预防前往非洲的旅行者和居民感染疟疾，并对整个社区进行免疫接种 传播并消除非洲重点地区的疟疾。未来5-10年，我们 旨在显着提高基于 PfSPZ 的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。我们提高保护广度的策略之一 疫苗将包括来自不同地理区域的其他 Pf 菌株，例如 Pf7G8（巴西）和 PfNF135.C10（柬埔寨）。除 PfNF54（西非菌株）外，在人类中评估的所有其他 Pf 菌株均 配子体生产者较差。提高 PfSPZ 大规模生产效率的方法 不同的地理区域和不断减少的重心将产生更多的可育数量 用于在蚊子中生产 PfSPZ 的每单位培养物的配子细胞。在自然界中，配子细胞发生发生 由于配子细胞发生相关的表观遗传抑制，仅存在于血液期寄生虫的一小部分中 基因。配子体定型的控制将为提高 PfSPZ 的生产提供有力的工具。一个 这个过程中的关键分子是主开关转录因子PfAP2-G，其表达 与给定 Pf 菌株产生的配子体百分比直接相关。删除该基因 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度表达该基因来减少 PfSPZ 制造 COG CRISPR-Cas9 基因编辑可修改 pfap2-g 3’-UTR 中的配对元件 (PE)。作为替代方案，我们 还将通过用组成型钙调蛋白替换 PfAP2-G 的启动子来缓解 PfAP2-G 的表观遗传沉默 我们开发的条件调节配子细胞诱导（开/关）系统中的启动子。我们会 从不同地理区域生成增强的 Pf 配子体生产系，进行比较和评估 具有增强的配子细胞发生的克隆，并选择具有最佳体外生长且维持高水平的克隆 无菌蚊子中传染性 PfSPZ 的流行率和强度。

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.