Engineering of genetically attenuated pre-erythrocytic Plasmodium parasites for cross-stage protective immunity

项目总结/摘要 研制有效的疟疾疫苗仍然是一个重要的全球卫生优先事项。基准 疟疾疫苗有效性的一个观察是，用完整的活疟原虫免疫， 蚊子叮咬产生子孢子，对疟原虫提供完全的无菌保护 动物模型中的感染和用疟原虫进行的控制人类疟疾感染（CHMI）试验 恶性疟原虫。最近的临床试验也证明了在免疫后对CHMI的完全保护。 人类志愿者与胃肠外给药减毒子孢子。这种保护水平还没有 与疟疾疫苗候选亚单位制剂相匹配。除了制造业的挑战， 活子孢子免疫的一个令人生畏的挑战性方面是通过完全免疫来确保安全性。 减毒，同时保持最佳免疫原性。有三种主要的方法论， 子孢子免疫：放射减毒子孢子（RAS），子孢子化学预防（CPS） 和遗传减毒寄生虫（GAP）。与CPS和RAS相反，GAP被良好的- 特征性的、一致的基因缺失，其允许控制肝脏期间寄生虫停滞的时间点， 阶段发展。这具有更大的安全性的潜力，但也具有上级功效，后者 通过发现晚期肝脏阶段的啮齿类疟疾GAP赋予持久的、上级的 红细胞前免疫和小鼠的跨阶段免疫。此外，我们最近设计了一个 P.恶性疟原虫早期肝脏阶段-通过三重基因缺失阻止GAP，首次显示完全 通过蚊子叮咬高剂量接种的人类志愿者的红细胞前衰减。这里我们将 在这些成功的基础上，提出设计和设计下一代GAP。我们就来 双管齐下的方法：在目标1中，我们将修改早期肝脏阶段阻滞GAP， 在约氏疟原虫啮齿动物疟疾模型和恶性疟原虫中的减毒，以表达血液阶段和 通过转基因工程获得配子体抗原。在目标2中，我们将产生新的晚期肝脏阶段阻滞， 通过组合缺失影响肝脏晚期发育的基因完全减弱GAP，然后 通过转基因增强其与血液阶段和配子体抗原的跨阶段保护能力 工程.最后，在目标3中，我们将评估所有新型GAP诱导完全灭菌的能力 红细胞前免疫、对无性血液阶段以及有性阶段的跨阶段免疫 传播阻断免疫力我们还将阐明免疫机制的保护所产生的 新GAP免疫。该应用程序的最终交付将是下一代P。 恶性疟原虫GAP是安全的，并根据令人信服的实验证据预测， 对红细胞前期感染、无性血阶段寄生虫血症的保护， 传播给蚊子媒介。

PROJECT SUMMARY/ABSTRACT The development of an effective malaria vaccine remains an important global health priority. A benchmark of malaria vaccine efficacy has been the observation that immunization with whole, live Plasmodium sporozoites that are adminstered by mosquito bite, confers complete sterile protection to malaria parasite infection in animal models and controlled human malaria infection (CHMI) trials with Plasmodium falciparum. Recent clinical trials also demonstrated complete protection to CHMI after immunization of human volunteers with parenterally administered attenuated sporozoites. This level of protection has yet to be matched by malaria vaccine candidate subunit formulations. Next to challenges in manufacturing, a formidably challenging aspect of live sporozoite immunization is ensuring safety by means of complete attenuation, while maintaining optimal immunogenicity. There are three main methodologies for live sporozoite immunization: radiation-attenuated sporozoites (RAS), chemoprophylaxis with sporozoites (CPS) and genetically attenuated parasites (GAP). In contrast to CPS and RAS, GAP are attenuated with well- characterized, consistent gene deletions that allow the control of the time point of parasite arrest during liver stage development. This has the potential for greater safety but also for superior efficacy, the latter demonstrated by the finding that late liver stage-arresting rodent malaria GAPs confer long lasting, superior pre-erythrocytic immunity and cross-stage immunity in mice. Furthermore, we have recently engineered a P. falciparum early liver stage-arresting GAP by triple gene deletion that showed for the first time complete pre-erythrocytic attenuation in human volunteers at high dose inoculation by mosquito bite. Here we will build on these successes and propose to design and engineer the next generation of GAP. We will take a two-pronged approach: In Aim 1 we will modify early liver stage-arresting GAP, that show complete attenuation in the P. yoelii rodent malaria model and in P. falciparum, to express blood stage and gametocyte antigens via transgene engineering. In Aim 2 we will generate novel late liver stage-arresting-, fully attenuated GAPs by combinatorial deletion of genes that affect late liver stage development, then augment their cross-stage protective capacity with blood stage and gametocyte antigens via transgene engineering. Finally in Aim 3, we will evaluate the capacity of all novel GAPs to induce completely sterilizing pre-erythrocytic immunity, cross-stage immunity to asexual blood stages as well as sexual stage transmision blocking immunity. We will also elucidate immune mechanisms of protection engendered by immunization with novel GAPs. The ultimate deliverable of this aplication will be a next generation P. falciparum GAP that is safe and is predicted, based on convincing experimental evidence, to afford protection against pre-erythrocytic infection, asexual blood stage parasitemia and will also prevent parasite transmission to the mosquito vector.