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

描述(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，目前尚不清楚的是抗疟疾CD8+T细胞在体内的激发方式。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)，辐射减弱子孢子(IrSpz)才能在猴子和小鼠的肝脏中诱导强大的疟疾特异性CD8+T细胞反应，并在小鼠中提供抗疟疾保护，因此这个悬而未决的问题尤为突出。因此，疫苗载体的性质以及疫苗接种的途径似乎会影响体内抗疟疾CD8+T细胞的诱导方式。这项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来源于约氏疟原虫环子孢子虫(PYCS)蛋白，是迄今为止已知的唯一一个CD8+表位，通过H-2Kd分子在小鼠中介导对约氏疟原虫感染的“保护”。因此，为了达到我们的总体目标，我们建立了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，分别使用CD11c启动子、huCD68启动子或白蛋白启动子。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、保护性的抗疟疾免疫。这些MHC-I-KD转基因小鼠将作为阳性对照。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，然后用氯喹治疗，通过不同的途径。在目标1中，我们将检测在每组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。在目标2中，我们将用活的疟疾寄生虫攻击这些免疫的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，并过继地将它们转移到未接种的MHC-I-KD TG小鼠，然后攻击疟疾，从而确定哪些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抗原特异的CD8+T细胞，并过继地将它们转移到NA-MHC-I-KD TG小鼠，随后进行疟疾攻击，以确定目标4中CD8+T细胞的保护能力。总体而言，我们相信，识别抗疟疾“保护性”CD8+T细胞的诱导机制可能最终导致针对人类疟疾的有效T细胞疫苗的极大改进设计。

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.