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

使用恶性疟原虫血期营养缺陷型的减毒疟疾子孢子疫苗

• 批准号: 8607502
• 负责人: STEPHEN Lev HOFFMAN
• 金额: $ 30万
• 依托单位: SANARIA, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8607502
• 关键词: 1-deoxy-2-pentulose; Adverse effects; Anabolism; Anopheles Genus; Antigens; Antimalarials; Area; Attenuated; Azithromycin; Biodistribution; Biological Assay; Biological Models; Bite; Blood; Chemoprophylaxis; Chloroquine; Clinical Trials; Culicidae; Cyclic GMP; Development; Diet; Disease; Dose; England; Enzymes; Epitopes; Erythrocytes; Excision; Exposure to; Falciparum Malaria; Female; Gene Targeting; Generations; Genes; Genome; Germany; Growth; Growth and Development function; Hepatocyte; Human; Immune system; Immunity; Immunization; In Vitro; Infection; Knock-out; Liver; Maintenance; Malaria; Malaria Vaccines; Mali; Maryland; Mediating; Methods; Midgut; Modeling; Monitor; Mus; Netherlands; Oocysts; Parasites; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Phase I Clinical Trials; Plasmodium falciparum; Process; Production; Proteins; Quality Control; Risk; Rodent; Rodent Model; Safety; Salivary Glands; Sporozoite vaccine; Sporozoites; Staging; Supplementation; System; Tanzania; Technology; Testing; Time; Tissue Model; Tissues; Toxicology; Vaccines; Validation; Zinc Fingers; asexual; base; controlled release; cost; disease transmission; drug metabolism; feeding; fosmidomycin; immunogenicity; improved; inorganic phosphate; isopentenyl pyrophosphate; isoprenoid; killings; mutant; nuclease; pre-clinical; prevent; protective efficacy; public health relevance; tool; transmission process; vaccine candidate; vaccine safety; volunteer; zinc finger nuclease

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.

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 3 年的努力，最有效的亚单位疟疾疫苗仅在最后一次接种后 2 周内提供 50% 的感染保护，在 5 个月内提供 22% 的保护。 PfSPZ 是唯一能够诱导针对 Pf 的持续（至少 10-28 个月）、高水平（>90%）保护的免疫原。几十年来，焦点一直集中在辐照 (irr) PfSPZ 上。然而，最近，早期/中期肝脏发育所需基因被破坏的基因减毒 SPZ 在小鼠中显示出保护作用。此外，志愿者通过携带完全传染性 PfSPZ 的蚊子叮咬进行免疫，并服用氯喹来消除血液期寄生虫，在暴露于比携带 irrPfSPZ 的蚊子少 20 倍的感染 PfSPZ 的蚊子后，获得了针对肝期寄生虫的保护性免疫力。这种效率的提高可能是由于在肝脏中/晚期阶段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（包括与氯喹联合使用的 PfSPZ）的临床试验正在进行或计划进行。然而，如果不再需要抗疟药物那就太理想了。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来杀死肝脏晚期的寄生虫，在诱导保护性免疫方面比氯喹更有效。我们建议通过使用缺乏仅对晚期肝脏和无性血液阶段的发育至关重要的基因的Pf基因减毒菌株来消除对抗疟药物的需求并提高诱导保护性免疫的效率。使用新开发的、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端酶脱氧木酮糖5-磷酸还原异构酶（DXR）和甲基赤藓糖醇磷酸胞苷基转移酶（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，从而产生必需的代谢物异戊烯基二磷酸 (IPP)。 DXR 对于 Pf 血期生长至关重要。通过补充 IPP 可以逆转对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。将选择与野生型寄生虫相比产生可接受数量的配子体和 PfSPZ 的敲除克隆，并且 Sanaria 将产生纯化的、冷冻保存的 Pf?dxr+?lspD SPZ。对肝细胞和新肝组织模型的研究将使我们能够检验以下假设：Pf?dxr+?lspD 寄生虫在肝阶段发育后期（当顶端质体最活跃时）具有深刻的、IPP 依赖性的发育停滞。这些研究将建立一种候选疫苗，该疫苗具有最佳的免疫原性和关键的安全特性，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。