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

描述(由申请人提供)：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，这种疫苗可以完全预防感染。到目前为止，开发这样一种全球使用的疫苗的努力还没有成功。然而，用编码恶性疟原虫环子孢子蛋白的RTS、S/AS02亚单位疫苗接种后，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，减毒活疟原虫在小鼠和人类中都能产生完全和持久的CD8+细胞毒性T淋巴细胞(CTL)介导的保护，但这种疫苗制剂在现场部署不容易。对CTL靶向抗原的保护作用表明，CTL靶向的细胞将成为理想的亚单位疫苗抗原。以前，在整个生物体范围内寻找这样的靶点是不可能的，因为减毒寄生虫疫苗包含数千个不同的免疫靶点，导致复杂的多特异性CTL反应--直到现在，还没有能够在如此复杂的反应中识别离散抗原靶点的方法。为了解决这个问题，我将使用一种高通量的方法来有效地从疟疾暴露的小鼠中筛选数千个针对CTL的候选靶标，以便破译对疟疾具有免疫力的关键CTL反应。我假设保护性CTL反应包括广泛的抗原特异性，包括在肝脏和红细胞阶段表达的寄生虫蛋白。利用这种高通量微基因驱动的筛选方法，可以快速识别这些靶抗原。在具体目标1中，我将把这种方法与小鼠不同的疫苗接种模型结合起来，以评估CTL的动力学和多样性，并识别与保护相关的同源靶抗原。我还将使用补充的实验方法和电子计算机方法来指导重点候选目标库的开发。在具体目标2中，我将评估感染疟疾的红细胞对CTL的交叉激发，并确定这些靶点如何引发跨期CTL免疫。在这项工作中定义的免疫靶标稍后可以用于合理地测试来自疟疾暴露的人类的CTL，以定义人类的多特异性保护性CTL谱系。拟议的方法将成为确定保护性CTL目标的强大范例，并可普遍应用于疟疾和其他感染。此应用程序针对PA-10-059(K08职业发展奖)，旨在确保出色的指导、强大的机构支持和成功的合作。 叙述全球根除疟疾需要一种有效的抗感染疟疾疫苗。开发这样一种疫苗需要从疟疾寄生虫表达的数千种蛋白质中确定不同的抗原靶标。在这里，我们建议研究不同免疫方法在小鼠身上诱导的CTL，并通过高通量筛选技术和其他尖端方法分析CTL反应来识别保护性抗原，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.