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

项目摘要/摘要 恶性疟原虫全红细胞前(子孢子期和肝期)免疫 在人体临床试验中，疫苗具有灭菌免疫力。复制不足的疫苗，如辐射- 减毒的子孢子以子孢子的形式感染肝脏，但不发育为肝期裂殖体。复制- 然而，合格的疫苗会感染肝脏，并以组织裂殖体的形式复制。有多条证据表明 疟疾的动物模型已经表明，这种保护依赖于抗原特异性的CD8 T细胞 识别肝脏阶段感染的肝细胞，导致它们被消除。具有复制能力的寄生虫 接种疫苗赋予了对感染的卓越持久的灭菌免疫力，这似乎是在动物身上。 模型，与更广泛和更好的CD8 T细胞反应相关。然而，在很大程度上仍不清楚是如何做到的 整个减毒寄生虫疫苗独特的分子细胞生物学特性导致了 保护性CD8 T细胞的启动和同等重要，肝期抗原直接由 野生型肝脏阶段感染的肝细胞是疫苗诱导的保护性CD8 T细胞的目标。我们会 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 肝期感染的肝细胞导致肝期抗原与抗原的最佳交叉呈递 将细胞呈递给CD8T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，无法进行机械化的宿主研究。我们还将确定什么时候 野生型肝期发育点感染的肝细胞最容易受到效应CD8 T细胞的影响 居间淘汰。与此相一致，我们将确定动态肝脏阶段的转录本和蛋白质组 在整个发育过程中，向下选择最容易进入肝细胞的肝期蛋白的子集 加工和MHC I类限制性多肽呈递。在目标2中，我们将直接确定MHC类 Py和Pf的I多肽出现在感染的肝细胞上，特别是在最脆弱的时间点 并测试它们与寄生虫疫苗诱导的整个CD8 T细胞的反应性。然后我们将测试反应性表位，如 以及非反应性表位(隐蔽表位)作为载体亚单位疫苗在小鼠体内。如目标1所示，机械论 测试不能在PF中进行，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，我们将 基因工程的终极PF复制能力的寄生虫株是通过基因缺失和 寄生虫来源的显性负性转基因，也将过度表达保护性CD8 T细胞表位， 以肝脏最脆弱的阶段为目标。因此，我们的项目将多管齐下 开发下一代前红细胞疫苗，包括矢量化亚单位疫苗候选者和 完整的基因减毒寄生虫候选疫苗，旨在产生最佳和持久的保护 CD8T细胞对PF感染的免疫应答

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.