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

疟疾造成约445 000人死亡，尽管有目前的控制方法，仍有2亿多人感染， 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， 恶性疟原虫是造成这些死亡的主要寄生虫， 这是一个里程碑，拯救了数百万人的生命。开发这种疫苗需要可靠和具有成本效益的 恶性疟原虫子孢子的产生。不幸的是，这在技术上或 经济地分离足够数量的这些子孢子以用于从感染者中广泛接种疫苗 蚊子。因此，另一种制备这些子孢子的方法对疫苗开发至关重要 和生产. MalarVx，Inc.已经证明了在实用的、成本有效的培养物中生长子孢子的能力 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所的布兰登怀尔德博士合作， 6（NAMRU 6）在利马，秘鲁，我们建议证明，恶性疟原虫子孢子产生在 体外分离的恶性疟原虫子孢子在生物学上等同于从 被感染的蚊子我们将测试体外产生的子孢子的感染性和体内发育， 人源化小鼠模型，和2）非人灵长类动物中的免疫原性。这些研究将使用野生的- NF 54型GFP-荧光素酶（GFP-Luc）报告菌株和NF 54 GAP 3 KO，一种遗传减毒寄生虫 目前正在进行临床开发作为候选疫苗的菌株。 为了检验等效性，我们将研究子孢子中表达的蛋白质标记，包括子孢子的主要表面。 蛋白CSP以及TRAP和mTIP。我们还将分析子孢子的功能，包括滑行运动， 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 肝细胞侵入、穿越和感染。 最后，NAMRU 6的怀尔德博士将监督对培养的子孢子的免疫原性的测试， 澳洲猕猴Aotus是唯一有免疫能力的动物（人类除外），可以完全感染 恶性疟原虫和间日疟原虫这两种主要的人类疟疾寄生虫。 这项工作将支持Aotus模型的进一步发展，用于检测WSV的安全性、免疫原性， 和抗多种疟原虫菌株和物种的功效。 在SBIR的第二阶段，我们将1）增加我们的培养系统和SPZ纯化的规模 工艺，2）优化冷冻保存，3）进行额外的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.