LENTIVIRUS TRANSDUCED DENDRITIC CELLS FOR HIV VACCINES

描述（改编自申请人摘要）：研究者将 专注于开发基于人类树突状细胞（DC）的疫苗策略， 诱导病毒特异性细胞毒性T淋巴细胞（CTL）免疫。 小说 将构建复制缺陷型慢病毒（HIV-1）载体， 将抗原表位递送到非复制的成熟和 不成熟的DC 具体目标1将决定是否完全功能 从血液单核细胞培养的“成熟”DC（mo-DC）将产生HIV特异性的 CD8+ CTL。 短期培养的mo-DC可以很容易地产生与at 95%的同质性。 三种env/nef缺失的HIV-1载体 VSV（G）的伪类型化将被评估。 他们的LTR被证明是 在mo-DC中高效转录。 为了产生HIV特异性CTL应答， 来自HLA-A2.1健康供体的CD 8 + T细胞将用 病毒转导的mo-DC在体外，使用为生产建立的条件 大于10 E8肽特异性CTL。 CTL的特异性为 通过裂解病毒感染的A2.1-Jurkat细胞来测量。 反应性 已知gag和pol的A2-1限制性表位也将用 A2- 1-用适当的肽脉冲的Jurkat细胞。 如果病毒特异性 CTL反应性不针对已知的表位，研究人员将 通过一组纳米体（“模拟表位”）鉴定新表位， gag或pol蛋白的免疫原性结构域。 冷冻转导的mo-DC可 用于重复体内免疫，或产生用于过继性T细胞的CTL 细胞疗法 特异性目标2将确定新鲜（未培养）DC 并且定向DC前体是有用的转导靶。 新鲜 来自G-CSF动员的外周血的成熟和未成熟DC群 将被感染，以预测使用慢病毒载体的可行性， 体内免疫。 特别是，研究人员将监测是否 转导阻碍了抗原呈递功能的发展， 未成熟DC，如通过其产生HIV特异性CTL的能力所测量的， 适当的共刺激分子和辅助分子的表达。 如果 必要时，研究人员将把病毒载体 细胞因子如flt 3配体（FL）或IL-4，以促进 新鲜的DC。 具体目标3将验证体内转导是可行的 在小鼠模型中。 研究人员将首先用 用具有CMV启动子的HIV载体转导的同基因DC，并测量 脾脏和淋巴结中的病毒特异性CTL活性。 用于概念验证 研究人员将在体内免疫小鼠， 在每天注射FL后， 体内DC前体的数量。

DESCRIPTION (Adapted from applicant's abstract): The investigators will focus on developing a human dendritic cell (DC)-based vaccine strategy to induce virus-specific cytotoxic T lymphocyte (CTL) immunity. Novel replication-defective lentiviral (HIV-1) vectors will be constructed to deliver antigenic epitopes into nonreplicating populations of mature and immature DC. Specific Aim 1 will determine whether fully functional "mature" DC cultured from blood monocytes (mo-DC) will generate HIV-specific CD8+ CTL. Short-term cultures of mo-DC can be easily generated with at least 95 percent homogeneity. Three env/nef deleted HIV-1 vectors pseudotyped with VSV(G) will be evaluated. Their LTR were shown to be productively transcribed in mo-DC. To generate a HIV-specific CTL response, CD8+ T cells from HLA-A2.1 healthy donors will be primed with virus-transduced mo-DC in vitro, using conditions established for production of greater than 10E8 peptide-specific CTL. Specificity of the CTL will be measured by lysis of virus-infected A2.1-Jurkat cells. Reactivities to known A2-1-restricted epitopes of gag and pol also will be assessed with A2.-1-Jurkat cells pulsed with appropriate peptides. If the virus-specific CTL reactivity was not directed to known epitopes, the investigators will identify the novel epitopes by a panel of nanomers ("mimotopes") encoding the immunogenic domains of gag or pol proteins. Frozen transduced mo-DC may be used for repeat in vivo immunizations, or to generate CTL for adoptive T cell therapy. Specific Aim 2 will determine whether fresh (uncultured) DC and committed DC precursors are useful targets for transduction. Fresh mature and immature DC populations from G-CSF-mobilized peripheral blood will be infected, to predict the feasibility of using lentiviral vectors to immunize in vivo. In particular, the investigators will monitor whether transduction impedes the development of antigen presenting functions of immature DC as measured by their ability to generate HIV-specific CTL and expression of appropriate costimulatory and accessory molecules. If necessary, the investigators will incorporate into the viral vectors cytokines such as flt3 ligand (FL) or IL-4, to facilitate maturation of fresh DC. Specific Aim 3 will verify that in vivo transduction is feasible in a murine model. The investigators will first immunize mice with syngeneic DC transduced by the HIV vector with the CMV promoter, and measure virus-specific CTL activity in spleen and lymph nodes. For proof of concept of in vivo transduction, the investigators will immunize mice in vivo with the vector, after daily injections of FL designed to dramatically increase the number of DC precursors in vivo.