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，因此需要研制有效的疫苗。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，抗疟疾的CD8+ T细胞是如何在体内被激发的仍是未知的。鉴于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，并在小鼠中提供抗疟疾保护，因此这个未解决的问题尤为突出。因此，疫苗载体的性质以及疫苗接种途径似乎会影响体内“保护性”抗疟疾CD8+ T细胞的诱导模式。该建议的总体目标是确定体内诱导抗疟疾CD8+ T细胞的机制。SYVPSAEQI来源于P. yoelii环孢子子（PyCS）蛋白，是迄今为止唯一已知的介导对小鼠P. yoelii感染的“保护”的CD8+表位，并通过H-2Kd分子呈现。因此，为了实现我们的总体目标，我们通过使用CD11c启动子、huCD68启动子或白蛋白启动子，分别产生了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。我们还培养了MHC-I-Kd Tg小鼠，在MHC-I启动子下表达Kd分子，我们可以在其中诱导有效的，保护性的抗疟疾免疫，依赖于PyCS蛋白和CD8+ T细胞。这些MHC-I-Kd - Tg小鼠将作为阳性对照。在这项研究中，我们将用疟疾疫苗免疫Kd - Tg小鼠，包括表达PyCS抗原、IrPySpz或活PySpz的腺病毒，然后用氯喹治疗，通过不同的途径。我们将在Aim 1中确定每组Kd - Tg小鼠中诱导的PyCS抗原特异性CD8+ t细胞反应的数量、质量和持久性。在第二阶段，我们将用活的疟疾寄生虫刺激这些免疫的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Aim 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，并将其移入无MHC-I-Kd - Tg小鼠，然后进行疟疾攻击，来确定哪些表达Kd的细胞诱导了保护性抗疟疾免疫。最后，我们将从免疫的各种Kd - Tg小鼠中分离PyCS抗原特异性CD8+ T细胞，并过继性地将其转移到无MHC-I-Kd - Tg小鼠中，随后进行疟疾攻击，以确定CD8+ T细胞在Aim 4中的保护能力。总之，我们相信，识别抗疟疾“保护性”CD8+ T细胞的诱导机制，最终将大大改进基于T细胞的抗人类疟疾疫苗的设计。

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.