Mechanisms of induction of protective anti-malarial CD8+ T Cells

描述（由申请人提供）：疟疾仍然是世界上最具破坏性的传染病之一，这凸显了开发有效疫苗的必要性。目前针对肝脏阶段的候选疟疾疫苗可诱导 CD8 T 细胞介导的保护。然而，目前尚不清楚抗疟疾 CD8 T 细胞在体内的产生方式。这个悬而未决的问题尤其突出，因为最近的一项研究表明，只有通过静脉注射（而不是其他途径），辐射减毒子孢子（IrSpz）才能在猴子和小鼠的肝脏中诱导有效的疟疾特异性 CD8 T 细胞反应，并为小鼠提供抗疟疾保护。因此，疫苗载体的性质以及疫苗接种途径似乎影响体内“保护性”抗疟疾 CD8 T 细胞的诱导模式。该提案的总体目标是确定体内诱导抗疟疾 CD8 T 细胞的机制。 SYVPSAEQI 源自约氏疟原虫环子孢子 (PyCS) 蛋白，是迄今为止唯一已知的介导小鼠免受约氏疟原虫感染“保护”的 CD8 表位，由 H-2Kd 分子呈递。因此，为了实现我们的总体目标，我们分别使用 CD11c 启动子、huCD68 启动子或白蛋白启动子生成了 C57BL/6 转基因 (Tg) 小鼠，其中 Kd 分子仅在树突状细胞 (DC) (CD11c-Kd)、巨噬细胞 (huCD68-Kd) 或肝细胞 (Alb-Kd) 上表达。我们还培育了在 MHC-I 启动子下表达 Kd 分子的 MHC-I-Kd Tg 小鼠，在其中我们可以诱导依赖于 PyCS 蛋白和 CD8 T 细胞的有效、保护性抗疟疾免疫。这些 MHC-I-Kd Tg 小鼠将用作阳性对照。在拟议的研究中，我们将用疟疾疫苗（包括表达 PyCS 抗原的腺病毒、IrPySpz 或活 PySpz）对 Kd Tg 小鼠进行免疫，然后通过不同途径用氯喹治疗。我们将确定目标 1 中每组 Kd Tg 小鼠诱导的 PyCS 抗原特异性 CD8 T 细胞反应的数量、质量和持久性。在目标 2 中，我们将用活疟原虫攻击这些免疫的 Kd Tg 小鼠，以确定体内诱导的保护性免疫的水平和持久性。在目标 3 中，我们将通过从免疫的各种 Kd Tg 小鼠中分离这些 Kd 细胞，并将其过继转移至初始 MHC-I-Kd Tg 小鼠，然后进行疟疾攻击，确定哪些 Kd 表达细胞诱导保护性抗疟疾免疫。最后，我们将从免疫的各种 Kd Tg 小鼠中分离出 PyCS 抗原特异性 CD8 T 细胞，并将其过继转移到幼稚 MHC-I-Kd Tg 小鼠中，然后进行疟疾攻击，以确定目标 4 中 CD8 T 细胞的保护能力。总体而言，我们相信抗疟疾“保护性”CD8 T 细胞的诱导机制的识别最终可能导致针对人类的有效 T 细胞疫苗的设计得到极大改进。疟疾。

DESCRIPTION (provided by applicant): Malaria remains one of the most devastating infectious diseases of the world, underscoring the need to develop effective vaccines. The current candidate malaria vaccines against the liver stages induce CD8+ T- cell-mediated protection. However, what remains unknown is the manner in which the anti-malarial CD8+ T cells are elicited in vivo. This unanswered question is particularly prominent in view of a very recent study showing that only through intravenous administration (and no other routes) do radiation-attenuated sporozoites (IrSpz) induce a potent malaria-specific CD8+ T-cell response in the livers of monkeys and of mice and provide anti-malarial protection in mice. Therefore, it appears that the nature of vaccine vectors, as well as the routes of vaccination, influences the mode of induction of "protective" anti-malarial CD8+ T cells in vivo. The overall aim of this proposal is to determine the mechanisms of in vivo induction of anti-malarial CD8+ T cells. SYVPSAEQI, derived from the P. yoelii circumsporozoite (PyCS) protein, is to date the only known CD8+ epitope that mediates "protection" against P. yoelii infection in mice and is presented by an H-2Kd molecule. Therefore, in addressing our overall goal, we have generated C57BL/6 transgenic (Tg) mice, in which Kd molecule is expressed only on dendritic cell (DC) (CD11c-Kd), macrophage (huCD68-Kd), or hepatocyte (Alb- Kd), by using CD11c promoter, huCD68 promoter, or albumin promoter, respectively. We have also generated MHC-I-Kd Tg mice that express a Kd molecule under the MHC-I promoter, in which we could induce a potent, protective anti-malarial immunity, dependent on both the PyCS protein and CD8+ T cells. These MHC-I-Kd Tg mice will be used as a positive control. In the proposed study, we will immunize the Kd Tg mice with malaria vaccines, including an adenovirus expressing the PyCS antigen, IrPySpz, or live PySpz followed by treatment with chloroquine, by different routes. We will determine the quantity, quality, and durability of PyCS antigen- specific CD8+ T-cell response induced in each group of Kd Tg mice in Aim 1. In Aim 2, we will challenge these immunized Kd Tg mice with live malaria parasites to determine the level and persistence of protective immunity induced in vivo. In Aim 3, we will determine which Kd-expressing cells induce the protective anti-malarial immunity by isolating these Kd+ cells from immunized, various Kd Tg mice, and adoptively transferring them to na¿ve MHC-I-Kd Tg mice, followed by a malaria challenge. Finally, we will isolate PyCS antigen-specific CD8+ T cells from immunized, various Kd Tg mice and adoptively transfer them to na¿ve MHC-I-Kd Tg mice, followed by a malaria challenge, to determine the protective capacity of the CD8+ T cells in Aim 4. Overall, we believe that the identification of the induction mechanisms of anti-malarial "protective" CD8+ T cells could ultimately lead to the vastly improved designs of potent T-cell-based vaccines against human malaria.