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

描述（由申请方提供）：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个月仅提供50%的感染保护。PfSPZ是唯一的免疫原，诱导持续（至少10-28个月），高水平（>90%）的保护对Pf.For几十年来的焦点一直是照射（irr）PfSPZ。然而，最近，在早期/中期肝脏发育所需的基因中破坏的遗传减毒SPZ在小鼠中显示出保护作用。此外，通过携带完全传染性PfSPZ的蚊子叮咬免疫的志愿者，并给予氯喹以消除血液期寄生虫，在暴露于比携带PfSPZ的蚊子少20倍的PfSPZ感染的蚊子后获得针对肝脏期寄生虫的保护性免疫。这种提高的效率可能是由于在中期/晚期肝脏阶段期间呈递给免疫系统的寄生虫表位的丰度和多样性增加。PfSPZ疫苗的临床试验正在进行或计划进行，包括PfSPZ与氯喹联合给药。然而，如果消除对抗疟药物的需求，那将是理想的。此外，在啮齿动物疟疾模型中，药物阿奇霉素通过破坏顶质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。我们建议消除对抗疟药物的需求，并通过使用遗传减毒的Pf菌株来提高诱导保护性免疫的效率，所述Pf菌株缺乏对于仅晚期肝脏和无性血液阶段的发展至关重要的基因。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，我们将删除分别编码顶质体酶脱氧木酮糖5-磷酸还原异构酶（DXR）和甲基异丙基磷酸胞苷转移酶（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，导致产生必需代谢物异戊烯基二磷酸（IPP）。DXR对Pf血液期生长至关重要。顶质体发育和类异戊二烯生物合成的抑制可以通过补充IPP来逆转，从而允许产生双敲除寄生虫。敲除克隆将被选择，产生可接受的配子母细胞和PfSPZ的数量相比，野生型寄生虫，和Sanaria将产生纯化，冷冻保存的Pf？dxr+？lspD SPZ。在肝细胞和一个新的肝组织模型的研究将使我们的假设，Pf？dxr+？lspD寄生虫在肝脏发育阶段后期具有深刻的IPP依赖性发育停滞，此时顶质体最活跃。这些研究将建立一种具有最佳免疫原性和关键安全性特征的候选疫苗，如果任何寄生虫从肝脏突破，则不能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.