Attenuated malaria sporozoite vaccine using a P. falciparum blood-stage auxotroph

描述（由申请人提供）：一种针对最致命的人类疟疾寄生虫恶性疟原虫（Pf）的无症状孢子子（SPZ）和肝脏阶段的高效疫苗，将是预防疟疾感染、疾病和传播的理想工具。经过30年的努力，最有效的亚单位疟疾疫苗在最后一次接种后2周仅提供50%的预防感染保护，5个月仅提供22%的保护。PfSPZ是唯一能诱导持续（至少10-28个月）、高水平（> - 90%）抗Pf的免疫原。几十年来，人们一直关注辐照（irr） PfSPZ。然而，最近，基因减弱的SPZ在早期/中期肝脏发育所需的基因中被破坏，在小鼠中显示出保护作用。此外，志愿者被携带完全感染性PfSPZ的蚊子叮咬后免疫，并给予氯喹以消除血期寄生虫，在接触感染PfSPZ的蚊子后，获得对肝期寄生虫的保护性免疫，比接触携带irrPfSPZ的蚊子少20倍。这种提高的效率可能是由于在中晚期肝脏阶段向免疫系统提供的寄生虫表位的丰度和多样性增加。基于PfSPZ的疫苗正在进行或计划进行临床试验，包括PfSPZ与氯喹一起施用。然而，如果消除了对抗疟疾药物的需求，那将是理想的。此外，在啮齿动物疟疾模型中，阿奇凝血素药物在诱导保护性免疫方面比氯喹更有效。阿奇凝血素通过破坏顶端质体功能，在肝脏晚期杀死寄生虫。我们建议通过使用一种基因减毒的Pf缺乏基因的菌株来消除对抗疟药物的需要，并提高诱导保护性免疫的效率，这些基因对晚期肝脏和无性血阶段的发展至关重要。利用一种基于定制锌指核酸酶的新开发的高效基因组编辑方法，我们将分别删除编码顶质体酶脱氧纤维素5-磷酸还原异构酶（dxr）和甲基赤藓糖醇磷酸胞基转移酶（IspD）的pfdxr和lspD基因。DXR催化类异戊二烯生物合成的第一步，随后是IspD，产生必需代谢物二磷酸异戊二烯（IPP）。DXR对Pf血期生长至关重要。通过补充IPP可以逆转顶质体发育和类异戊二烯生物合成的抑制，从而产生双敲除寄生虫。与野生型寄生虫相比，将选择产生可接受数量的配子体和PfSPZ的敲除克隆，Sanaria将产生纯化的，冷冻保存的Pf?dxr+？lspD SPZ。内肝细胞和新的肝组织模型的研究将使我们能够验证Pf?dxr+？lspD寄生虫在顶端质体最活跃的肝脏发育晚期有一个深刻的、依赖于ipp的发育停滞。这些研究将建立一种候选疫苗，具有最佳的免疫原性和一个关键的安全特征，即如果任何寄生虫从肝脏突破，则无法在红细胞中维持复制。

DESCRIPTION (provided by applicant): A highly effective vaccine that targets the asymptomatic sporozoite (SPZ) and liver stages of the most lethal human malaria parasite, Plasmodium falciparum (Pf), would be an ideal tool to prevent malaria infection, disease and transmission. After 3 decades of effort, the most effective subunit malaria vaccine only provides 50% protection against infection at 2 wks and 22% at 5 months after the last dose. PfSPZ are the only immunogens that induce sustained (at least 10-28 months), high level (>90%) protection against Pf. For decades the focus has been on irradiated (irr) PfSPZ. Recently, however, genetically attenuated SPZ disrupted in genes required for early/mid liver stage development have shown protection in mice. Furthermore, volunteers immunized by the bite of mosquitoes carrying fully infectious PfSPZ, and administered chloroquine to eliminate blood stage parasites, acquire protective immunity against liver stage parasites after exposure to 20 times fewer PfSPZ-infected mosquitoes than are required with mosquitoes carrying irrPfSPZ. This increased efficiency is likely due to increased abundance and diversity of parasite epitopes presented to the immune system during the mid/late liver stages. Clinical trials are underway or planned for PfSPZ-based vaccines, including PfSPZ administered with chloroquine. However, it would be ideal if the need for an antimalarial drug were eliminated. Furthermore, in a rodent malaria model the drug azithromcyin, which kills parasites at the late liver stage by disrupting apicoplast function, is more efficient than chloroquine in inducing protective immunity. We propose to eliminate the need for an antimalarial drug and improve the efficiency of induction of protective immunity by using a genetically attenuated strain of Pf lacking genes that are essential for development of only late liver and asexual blood stages. Using a newly developed, highly efficient method for genome editing based on customized zinc-finger nucleases, we will delete the Pf dxr and lspD genes that encode apicoplast enzymes deoxyxylulose 5-phosphate reductoisomerase (DXR) and methylerythritol phosphate cytidyltransferase (IspD), respectively. DXR catalyzes the 1st step in isoprenoid biosynthesis, followed by IspD, resulting in production of the essential metabolite isopentenyl diphosphate (IPP). DXR is essential for Pf blood stage growth. Inhibition of apicoplast development and isoprenoid biosynthesis can be reversed by supplementation with IPP allowing for generation of double knockout parasites. Knockout clones will be selected that generate acceptable numbers of gametocytes and PfSPZ when compared to wild type parasites, and Sanaria will produce purified, cryopreserved Pf?dxr+?lspD SPZ. Studies in hepatocytes and a new liver tissue model will enable our testing of the hypothesis that Pf?dxr+?lspD parasites have a profound, IPP-dependent, developmental arrest late during liver stage development when the apicoplast is most active. These studies will establish a vaccine candidate with optimal immunogenicity and a critical safety feature of being unable to sustain replication in erythrocytes if any parasites break through from the liver.