LANGERHANS CELL TARGETED GENETIC VACCINE AGAINST HIV1

描述：体液和细胞免疫反应均可由 编码外源抗原（Ag）的裸质粒DNA。 这 基因疫苗接种技术已被用于诱导免疫反应 包括HIV-1在内的传染性病原体。 在这些实验中， 启动子被用作转录调控单元，因此，导致了 基因产物的表达（即，Ag）在几乎所有细胞类型中。 我们 现在建议开发一种新的针对HIV-1的疫苗， 将gp 120基因表达到朗格汉斯细胞（LC）中，其具有所有 表型和功能特性所需的外源抗原呈递给 免疫幼稚T细胞。 事实上，LC被认为是一个关键的 在启动针对多种抗原的免疫应答中的作用，包括 传染性病原体 因此，我们假设抗HIV-1的疗效 通过将基因表达靶向LC可以显著改善疫苗。 我们进一步假设，这种疫苗将主要激活 MHC I类依赖性免疫臂（例如，cd 8 + T细胞介导 细胞毒性），因为Ag在LC内内源性产生。 我们有 还分离了一个独特的启动子，称为dectin-2启动子，并验证了其 在动物水平上将基因表达导向LC的能力。 我们还 观察到体外转染的LC系，当皮下注射时，在小鼠中， 诱导广泛的免疫应答（包括细胞毒性）。 因此我们 现在处于一个独特的位置来决定这种新的疫苗策略 可能适用于开发安全有效的疫苗 HIV-1 本研究的具体目的是：1）研究gp 120 mRNA的表达 和蛋白质。 我们将 通过基因枪递送pDec 2-gp 120质粒（含有编码 在dectin-2启动子控制下的gp 120序列）进入腹部 BALB/c小鼠皮肤，并测定其量、细胞和组织 gp 120 mRNA和蛋白表达的分布和时间过程。 2)到 表征由LC靶向的免疫应答诱导的gp 120特异性免疫应答 基因疫苗 将检查用pDec 2-gp 120接种的小鼠的 产生体液和T细胞介导的免疫应答， 不良影响 3)制定战略，提高 LC靶向基因疫苗。 有待测试的战略包括： 联合收割机与角化细胞靶向基因疫苗结合。 其中tPA-gp 120 基因（编码可溶形式的gp 120）在以下基因的控制下表达： 角蛋白-14启动子，用于激活II类依赖性臂，B）， 增加dectin-2启动子的转录活性，c）共递送 IL-12或IL-4基因以LC靶向方式用于选择性激活Th 1 或Th 2亚群，和d）在角质形成细胞靶向的细胞中递送GM-CSF基因， 以帮助LC成熟。 拟议的实验将， 最终，导致建立独特的疫苗策略， 最大限度地保护免受HIV-1感染，通过控制的幅度， 类（MHC I类与II类），和类型（Th 1与Th 2）， 免疫反应。

DESCRIPTION: Both humoral and cellular immune responses can be induced by inoculating naked plasmid DNA encoding a foreign antigen (Ag). This technology, genetic vaccination, has been used to induce immune responses against infectious pathogens, including HIV-1. In those experiments CMV promoter was used as a transcription regulatory unit, thus, leading to the expression of the gene products (i.e., Ag) in virtually all cell types. We now propose to develop a new class of vaccines against HIV-1, by directing the expression of gp120 gene to Langerhans cells (LC), which possess all phenotypic and functional properties required for presenting foreign Ag to immunologically naive T cells. In fact, LC are known to play a critical role in initiating immune responses against a wide variety of Ag, including infectious pathogens. Thus, we hypothesize that the efficacy of anti-HIV-1 vaccines can be improved significantly by targeting gene expression to LC. We hypothesize further that such a vaccine will activate predominantly the MHC class I-dependent arm of immunity (e.g., CD8+ T cell-mediated cytotoxicity), because the Ag is produced endogenously within LC. We have also isolated a unique promoter, termed dectin-2 promoter and validated its capacity to direct gene expression to LC in animal levels. We have also observed that an in vitro transfected LC line, when injected s.c. in mice, induces a wide array of immune responses (including cytotoxicity). Thus, we are now in a unique position to determine whether this new vaccine strategy is potentially applicable to the development of a safe and effective vaccine for HIV-1. Our specific aims are: 1) To study the expression of gp120 mRNA and protein after inoculation of LC-targeted genetic vaccine. We will deliver, by gene gun, the pDec2-gp120 plasmid (containing the coding sequence of gp120 under the control of dectin-2 promoter) into abdominal skin of BALB/c mice and determine the amount, cellular and tissue distributions, and time-course of gp120 mRNA and protein expression. 2) To characterize gp120-specific immune responses that are induced by LC-targeted genetic vaccine. Mice vaccinated with pDec2-gp120 will be examined for the generation of humoral and T cell-mediated immune responses and for potential adverse effects. 3) To establish strategies to improve the efficacy of LC-targeted genetic vaccine. The strategies to be tested include: a) to combine with keratinocyte-targeted genetic vaccine. in which the tPA-gp120 gene (encoding a soluble form of gp120) is expressed under the control of keratin-14 promoter, for activating the class II-dependent arm, b) to increase the transcriptional activity of dectin-2 promoter, c) to co-deliver IL-12 or IL-4 gene in an LC-targeted manner for selective activation of Th1 or Th2 subset, and d) to deliver GM-CSF gene in a keratinocyte-targeted manner to assist the maturation of LC. The proposed experiments will, ultimately, lead to the establishment of unique vaccine strategies to induce maximal protection against HIV-1 infection, by controlling the magnitude, the class (MHC class I versus class II), and the type (Th1 versus Th2) of immune responses.