Biologically informed design of CD8+ T cell-dependent pre-erythrocytic stage malaria vaccines

项目总结/摘要 用整个红细胞前（子孢子和肝阶段）恶性疟原虫（Pf）免疫 疫苗在人体临床试验中赋予了杀菌免疫力。复制缺陷疫苗，如辐射- 减毒的子孢子以子孢子的形式感染肝脏，但不发育成肝脏阶段的子孢子。复制- 然而，合格的疫苗感染肝脏并作为组织复制体进行复制。多条线索的证据 疟疾的动物模型显示，保护作用依赖于抗原特异性CD 8 + T细胞， 识别肝脏阶段感染的肝细胞，导致其消除。有复制能力的寄生虫 疫苗接种赋予针对感染的优越的上级持久的灭菌免疫，这似乎是，在动物中 模型，与更广泛和更好的CD 8 + T细胞反应相关。然而，在很大程度上仍然不知道如何 全减毒寄生虫疫苗的不同分子细胞生物学特征驱动了 启动保护性CD 8 + T细胞，同样重要的是，肝脏阶段抗原直接由 野生型肝脏阶段感染的肝细胞是疫苗诱导的保护性CD 8 + T细胞的靶。我们将 解决这些关键的知识差距。在目标1中，我们将确定死亡的不同时间点， 肝期感染肝细胞的最佳交叉呈递肝期抗原 向CD 8 + T细胞呈递细胞。为此，我们将使用约氏疟原虫（Py）啮齿动物疟疾模型来告知 Pf的疫苗设计，用其不能进行机理宿主研究。我们还将确定 野生型肝发育阶段感染的肝细胞最容易受到效应CD 8 + T细胞的影响， 介导的消除。与此同时，我们将确定动态肝脏阶段转录组和蛋白质组 在整个发育过程中，向下选择最倾向于肝内细胞凋亡的肝阶段蛋白质的子集， 加工和MHC I类限制性肽呈递。在目标2中，我们将直接确定MHC类别 Py和Pf的I肽组呈现在感染的肝细胞上，特别是在最高脆弱性的时间点 并测试它们与完整寄生虫疫苗诱导的CD 8 + T细胞的反应性。然后我们将测试反应性表位， 以及非反应性表位（隐蔽表位）作为小鼠中的载体亚单位疫苗。如目标1，机械性 Pf无法进行测试，因此我们将对Py进行研究，以指导我们的Pf工作。在目标3中，我们 基因工程改造最终的Pf复制能力寄生虫菌株，该菌株是用基因缺失构建的， 寄生虫来源的显性负转基因，并且还将过表达保护性CD 8 + T细胞表位， 针对肝脏最脆弱的阶段。因此，在多管齐下的方法，我们的项目将 开发下一代红细胞前疫苗，包括载体亚单位疫苗候选物， 全基因减毒寄生虫疫苗候选物，旨在产生最佳和持久的保护作用， 针对Pf感染的CD 8 + T细胞应答。

PROJECT SUMMARY/ABSTRACT Immunization with whole pre-erythrocytic (sporozoite and liver stage) Plasmodium falciparum (Pf) vaccines confers sterilizing immunity in human clinical trials. Replication-deficient vaccines, such as radiation- attenuated sporozoites infect the liver as sporozoites but do not develop into liver stage schizonts. Replication- competent vaccines, however, infect the liver and replicate as tissue schizonts. Multiple lines of evidence in animal models of malaria have shown that protection is dependent on antigen-specific CD8+ T cells that recognize liver stage-infected hepatocytes, leading to their elimination. Replication-competent parasite vaccination confers superior durable sterilizing immunity against infection, and this appears to be, in animal models, associated with broader and better CD8+ T cell responses. However, it remains largely unknown how the distinct molecular cell biological features of whole attenuated parasite vaccines drive differences in the priming of protective CD8+ T cells and of equal importance, which liver stage antigens are directly presented by wildtype liver stage-infected hepatocytes that are the targets of vaccine-elicited protective CD8+ T cells. We will address these critical knowledge gaps. In Aim 1, we will identify the distinct time points during which the demise of liver stage-infected hepatocytes results in optimal cross-presentation of liver stage antigens by antigen presenting cells to CD8+ T cells. For this, we will use the Plasmodium yoelii (Py) rodent malaria model to inform vaccine design of Pf, with which mechanistic host studies cannot be done. We will also determine at what time points of wildtype liver stage development infected hepatocytes are most vulnerable to effector CD8+ T cell- mediated elimination. In concert with this, we will determine dynamic liver stage transcriptomes and proteomes throughout development and down-select the subset of liver stage proteins most prone to intrahepatocytic processing and MHC class I-restricted peptide presentation. In Aim 2, we will directly determine the MHC class I peptidome of Py and Pf presented on infected hepatocytes, specifically at timepoints of highest vulnerability and test their reactivity with whole parasite-vaccine-elicited CD8+ T cells. We will then test reactive epitopes as well as nonreactive epitopes (covert epitopes) as vectored subunit vaccines in mice. As in Aim 1, mechanistic testing cannot be done in Pf and thus we will conduct studies of Py to guide our Pf work. In Aim 3, we will genetically engineer the ultimate Pf replication-competent parasite strain that is built with gene deletions and dominant negative transgenes of parasite origin and will also over-express protective CD8+ T cell epitopes that target liver stages at the point of their greatest vulnerability. Thus, in a multi-pronged approach our project will develop the next generation of pre-erythrocytic vaccines including both vectored subunit vaccine candidates and whole genetically attenuated parasite vaccine candidates, designed to generate optimal and durable protective CD8+ T cell responses against Pf infection.