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，强调需要开发有效的疫苗。目前针对肝脏阶段的候选疟疾疫苗诱导CD 8 + T细胞介导的保护。然而，仍然未知的是抗疟疾CD 8 + T细胞在体内被引发的方式。鉴于最近的一项研究表明，只有通过静脉内给药（而不是其他途径），放射减毒子孢子（IrSpz）才能在猴和小鼠的肝脏中诱导有效的疟疾特异性CD 8 + T细胞应答，并在小鼠中提供抗疟疾保护，这个未回答的问题尤为突出。因此，似乎疫苗载体的性质以及接种途径影响体内“保护性”抗疟疾CD 8 + T细胞的诱导模式。该提案的总体目标是确定抗疟疾CD 8 + T细胞的体内诱导机制。SYVPSAEQI来源于约氏疟原虫环子孢子（PyCS）蛋白，是迄今为止唯一已知的介导针对小鼠中约氏疟原虫感染的“保护”的CD 8+表位，并且由H-2Kd分子呈递。因此，为了实现我们的总体目标，我们已经产生了C57 BL/6转基因（Tg）小鼠，其中分别通过使用CD 11 c启动子、huCD 68启动子或白蛋白启动子，Kd分子仅在树突状细胞（DC）（CD 11 c-Kd）、巨噬细胞（huCD 68-Kd）或肝细胞（Alb-Kd）上表达。我们还产生了在MHC-I启动子下表达Kd分子的MHC-I-Kd Tg小鼠，其中我们可以诱导依赖于PyCS蛋白和CD 8 + T细胞的有效的保护性抗疟疾免疫。这些MHC-I-Kd Tg小鼠将用作阳性对照。在拟议的研究中，我们将用疟疾疫苗免疫Kd Tg小鼠，包括表达PyCS抗原的腺病毒IrPySpz或活PySpz，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小鼠中诱导的PyCS抗原特异性CD 8 + T细胞应答的数量、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，并将其过继转移到幼稚的MHC-I-Kd Tg小鼠，然后进行疟疾攻击，来确定哪些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PyCS抗原特异性CD 8 + 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CD 8 + T细胞的保护能力。总的来说，我们相信，抗疟疾“保护性”CD 8 + T细胞的诱导机制的鉴定可能最终导致针对人类疟疾的有效的基于T细胞的疫苗的设计得到极大的改进。

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.