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

描述(由申请人提供)：针对最致命的人类疟疾寄生虫恶性疟原虫(PF)的无症状子孢子(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周和5个月对感染的保护率分别为50%和22%。PfSPZ是唯一能诱导持续(至少10-28个月)、高水平(&gt；90%)的保护性肺炎的免疫原。几十年来，人们的注意力一直集中在辐照(辐射)PfSPZ上。然而，最近，基因减弱的SPZ破坏了肝脏早期/中期发育所需的基因，在小鼠中显示出保护作用。此外，志愿者通过携带完全感染PfSPZ的蚊子的叮咬进行免疫，并注射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力，所需数量比携带irrPfSPZ的蚊子所需的数量少20倍。这种效率的提高可能是由于在肝脏中后期呈现给免疫系统的寄生虫表位的丰度和多样性增加。基于PfSPZ的疫苗的临床试验正在进行中或计划进行，包括与氯喹一起注射的PfSPZ。然而，如果消除了对抗疟疾药物的需求，那将是最理想的。此外，在啮齿动物疟疾模型中，药物阿奇凝血素通过破坏质外体功能在肝脏晚期杀死寄生虫，在诱导保护性免疫方面比氯喹更有效。我们建议通过使用一种遗传减毒的PF菌株来消除抗疟疾药物的需要，并提高保护性免疫的诱导效率，该菌株缺乏仅对肝脏晚期和无性血液阶段的发育至关重要的基因。使用一种新开发的基于定制锌指核酸酶的高效基因组编辑方法，我们将分别删除编码质外体酶脱氧果糖5-磷酸还原异构酶(DXR)和甲基赤霉醇磷酸胞苷转移酶(IspD)的Pf dxr和lspD基因。DXR催化类异戊二烯生物合成的第一步，然后是IspD，产生必需的代谢物异戊烯基二磷酸(IPP)。DXR是PF血液期生长所必需的。通过添加IPP可以逆转对顶生质体发育和类异戊二烯生物合成的抑制，从而允许产生双敲除寄生虫。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，而Sanaria将产生纯化的、冷冻保存的Pf？dxr+？lspd SPZ。对肝细胞和一种新的肝组织模型的研究将使我们能够检验这一假设，即Pf？dxr+？lspd寄生虫在肝脏发育后期有一种深刻的、依赖于IPP的发育停滞，此时顶体最活跃。这些研究将建立一种具有最佳免疫原性和关键安全特征的候选疫苗，即如果有任何寄生虫从肝脏侵入，就无法维持红细胞内的复制。

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.