Identification of responding CD8+ T cells and novel protective epitopes following

描述（由申请方提供）：根除疟疾工作的成功取决于开发一种安全有效的疟疾疫苗，该疫苗可诱导完全的抗感染保护。迄今为止，开发这种疫苗供世界范围使用的努力尚未成功。然而，接种编码恶性疟原虫环子孢子蛋白的亚单位RTS，S/AS 02疫苗显著降低了非洲儿童因严重疟疾而死亡的人数，并首次证明了开发抗感染疟疾亚单位疫苗是可行的。我们知道减毒活疟原虫寄生虫在小鼠和人类中产生完全和持久的CD 8+细胞毒性T淋巴细胞（CTL）介导的保护作用，但这种疫苗制剂不容易在现场部署。保护对CTL靶向抗原的依赖性表明这些细胞的靶将成为理想的亚单位疫苗抗原。以前在整个生物体规模上寻找这样的靶标是不可能的，因为减毒寄生虫疫苗含有数千种不同的免疫靶标，导致复杂的多特异性CTL应答-直到现在还没有能够在这种复杂应答中鉴定离散抗原靶标的方法。为了解决这个问题，我将采用一种高通量的方法来有效地筛选成千上万的候选目标，从疟疾暴露的小鼠CTL，以破译关键的CTL反应，赋予免疫力，以对抗疟疾。我推测，保护性CTL反应包括广泛的抗原特异性，包括寄生虫蛋白表达在肝脏和红细胞阶段。这些靶抗原可以使用这种高通量小基因驱动的筛选方法快速鉴定。在具体目标1中，我将联合收割机将这种方法与小鼠中不同的疫苗接种模型相结合，以评估CTL的动力学和多样性，并确定与保护相关的同源靶抗原。我还将使用补充实验和计算机模拟方法来指导重点候选目标库的开发。在具体目标2中，我将评估疟疾感染的红细胞对CTL的交叉致敏，并确定这些靶点如何引发跨阶段CTL免疫。在这项工作中定义的免疫靶点可以在以后用于合理地测试来自疟疾暴露的人的CTL，以定义人类中的多特异性保护性CTL库。所提出的方法将作为一个强大的范例，用于确定保护性CTL目标，并可普遍适用于疟疾和其他感染。此应用程序地址PA-10-059（K 08职业发展奖），并精心制作，以确保优秀的指导，强大的机构支持和成功的合作。 全球根除疟疾需要一种有效的抗感染疟疾疫苗。开发这种疫苗需要从疟原虫表达的数千种蛋白质中鉴定出离散的抗原靶标。我们建议在这里研究不同的免疫方法诱导的小鼠CTL，并通过使用高通量筛选技术和其他尖端方法来分析CTL应答来识别保护性抗原，以加速疟疾疫苗的开发。

DESCRIPTION (provided by applicant): The success of malaria eradication efforts hinges on the development of a safe and efficacious malaria vaccine that induces complete protection against infection. To date, efforts to develop such a vaccine for worldwide use have been unsuccessful. However, vaccination with the subunit RTS,S/AS02 vaccine encoding the Plasmodium falciparum circumsporozoite protein significantly reduced deaths due to severe malaria in Africa children and demonstrated for the first time that development of an anti-infection malaria subunit vaccine is feasible. We know that live-attenuated Plasmodium parasites yield complete and long-lasting CD8+ cytotoxic T lymphocyte (CTL)-mediated protection in mice and humans, but such vaccine preparations are not easily deployed in the field. The dependence of protection on antigens targeted by CTLs indicates that the targets of such cells would make ideal subunit vaccine antigens. The search for such targets was previously impossible on a whole organism scale because attenuated parasite vaccines contain thousands of different immune targets leading to complex polyspecific CTL responses - until now there was no methodology capable of identifying discrete antigenic targets in the midst of such a complex response. To solve this problem, I will employ a high-throughput method to efficiently screen thousands of candidate targets against CTLs from malaria-exposed mice in order to decipher the key CTL responses that confer immunity against malaria. I hypothesize that protective CTL responses encompass a wide range of antigenic specificities and include parasite proteins expressed in both liver and erythrocyte stages. These target antigens can be rapidly identified using this high-throughput minigene-driven screening approach. In Specific Aim 1, I will combine this approach with different vaccination models in mice to assess the kinetics and diversity of CTLs and identify cognate target antigens associated with protection. I will also use complementary experimental and in silico approaches to guide development of a focused candidate target library. In Specific Aim 2, I will evaluate cross-priming of CTLs by malaria-infected erythrocytes and determine how such targets elicit cross-stage CTL immunity. Immune targets defined in this work can later be used to rationally test CTLs from malaria-exposed humans to define a polyspecific protective CTL repertoire in humans. The proposed approach will serve as a powerful paradigm for identifying protective CTL targets and can be generally applied to malaria and other infections. This application addresses PA-10-059 (K08 Career Development Award) and was crafted to ensure excellent mentorship, strong institutional support and successful collaborations. NARRATIVE Global eradication of malaria requires an efficacious anti-infection malaria vaccine. Development of such a vaccine requires the identification of discrete antigenic targets from among thousands of proteins expressed by malaria parasites. We propose here to study CTLs induced by different immunization approaches in mice and identify protective antigens by profiling CTL responses using high-throughput screening technologies and other cutting-edge approaches to accelerate malaria vaccine development.