Biological Equivalence of In Vitro Plasmodium falciparum Culture for Practical Malaria Vaccine Development

疟疾导致约44.5万人死亡，超过2亿人感染，尽管目前的控制方法， 包括蚊帐、杀虫剂、驱虫剂和抗疟疾药物。一种安全有效的疫苗 恶性疟原虫是造成大多数死亡的寄生虫，它将代表一种主要的公共卫生。 这是一个里程碑，拯救了数百万人的生命。开发这样一种疫苗需要可靠和成本效益高的 恶性疟原虫子孢子的产生。不幸的是，这在技术上或技术上都不可行 从经济上分离出足够数量的这些子孢子，以便广泛开展疫苗接种工作 蚊子。因此，另一种制造这些子孢子的方法对疫苗的开发至关重要。 和制作。 MalarVx，Inc.已经证明了在一种实用、成本效益高的培养条件下培养子孢子的能力 系统。这些子孢子紧密地概括了蚊子衍生的子孢子的性质，根据 全面的体外试验。与美国海军医学研究单位的布兰登·怀尔德博士合作- 6(NAMRU6)，我们建议证明恶性疟原虫子孢子产生于 在生物学上与从日本血吸虫唾液腺分离的恶性疟原虫子孢子相同 被感染的蚊子。我们将测试体外产生的子孢子1)在体内的传染性和发育 人源化小鼠模型；2)非人灵长类动物的免疫原性。这些研究将使用野生的- NF54 GFP-荧光素酶(GFP-Luc)报告菌株和NF54 GAP3KO，一种基因减毒的寄生虫 目前正在作为候选疫苗进行临床开发的菌株。 为了检验等价性，我们将研究在子孢子中表达的蛋白质标记，包括主表面。 蛋白CSP以及TRAP和mTIP。我们还将分析子孢子的功能，包括滑动运动和 肝细胞感染。我们将利用细胞培养方法和人源化肝脏嵌合小鼠模型来测试 肝细胞侵袭、遍历和感染。 最后，NAMRU6的怀尔德博士将监督培养的子孢子的免疫原性测试 奥图斯猴子。奥特斯是唯一可以完全感染的具有免疫能力的动物(人类除外) 恶性疟原虫和间日疟原虫是人类疟疾寄生虫的两个主要物种。 这项工作将支持Aotus模型的进一步开发，以测试WSV的安全性、免疫原性、 对多种品系和物种的疟原虫有一定的药效。 在这个SBIR的第二阶段，我们将1)扩大我们的培养系统和SPZ纯化的规模 过程，2)优化冷冻保存，以及3)执行额外的IND前研究。

Malaria kills about 445,000 people and infects over 200 million people despite current control methods, including bed nets, insecticides, insect repellants, and antimalarial drugs. A safe and effective vaccine against Plasmodium falciparum, the parasite responsible for most of these deaths, would represent a major public health milestone and save millions of lives. Development of such a vaccine will require reliable and cost-efficient production of Plasmodium falciparum sporozoites. Unfortunately, it is not feasible either technically or economically to isolate these sporozoites in sufficient numbers for widespread vaccination efforts from infected mosquitos. An alternative method of making these sporozoites is therefore essential to vaccine development and production. MalarVx, Inc. has demonstrated the capability of growing sporozoites in a practical, cost-effective culture system. These sporozoites closely recapitulate the properties of mosquito-derived sporozoites, according to comprehensive in vitro tests. In collaboration with Dr. Brandon Wilder at the U.S. Naval Medical Research Unit- 6 (NAMRU6) in Lima, Peru, we propose to demonstrate that Plasmodium falciparum sporozoites produced in vitro are biologically equivalent to Plasmodium falciparum sporozoites isolated from the salivary glands of infected mosquitoes. We will test the in vitro-produced sporozoites for 1) infectivity and development in vivo in a humanized mouse model, and 2) immunogenicity in non-human primates. These studies will use both the wild- type NF54 GFP-Luciferase (GFP-Luc) reporter strain and NF54 GAP3KO, a genetically attenuated parasite strain currently undergoing clinical development as a vaccine candidate. To test equivalence, we will study protein markers expressed in sporozoites, including the major surface protein CSP as well as TRAP and mTIP. We will also analyze sporozoite functions, including gliding motility and hepatocyte infection. We will utilize cell culture approaches and a humanized-liver chimeric mouse model to test hepatocyte invasion, traversal, and infection. Finally, Dr. Wilder at the NAMRU6 will supervise testing of the immunogenicity of cultured sporozoites in Aotus monkeys. Aotus are the only immune-competent animals (other than humans) that can be fully infected with both Plasmodium falciparum and Plasmodium vivax, the two major species of human malaria parasites. This work will support the further development of the Aotus model for testing of WSV safety, immunogenicity, and efficacy against multiple strains and species of Plasmodium. In Phase II of this SBIR, we will 1) increase the scale of our culturing system and the SPZ purification process, 2) optimize cryopreservation, and 3) perform additional pre-IND studies.