LENTIVIRUS TRANSDUCED DENDRITIC CELLS FOR HIV VACCINES

描述(改编自申请人的摘要)：调查人员将 专注于开发基于人树突状细胞(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 复制缺陷慢病毒(HIV-1)载体将被构建以 将抗原表位传递到非复制的成熟和 不成熟的DC。具体目标1将决定是否完全发挥作用 从血液单核细胞培养的成熟树突状细胞(mo-DC)将产生HIV特异性的 CD8+CTL。用At可以很容易地产生moDC的短期培养 至少95%的同质性。三个env/nef缺失的HIV-1载体 使用VSV(G)进行伪类型化将被评估。他们的LTR被证明是 在mo-DC中高效转录。为了产生HIV特异性的CTL反应， 来自HLA-A2.1健康捐赠者的CD8+T细胞将被 利用已建立的生产条件在体外转导病毒的mo-DC 大于10E8多肽的特异性CTL。CTL的特异性将是 通过病毒感染的A2.1-Jurkat细胞裂解来衡量。反应性 已知的A2-1限制性的GAG和POL表位也将通过 A2.-1-Jurkat细胞，用适当的多肽冲击。如果病毒特定 CTL反应不是针对已知表位的，调查人员将 通过一组纳米分子(“模拟表位”)编码识别新的表位 GAG或POL蛋白的免疫原域。冷冻换能式MO-DC可以 用于重复体内免疫，或用于产生过继T细胞的CTL 细胞疗法。具体目标2将决定新鲜(未培养的)DC 表达的DC前体是有用的转导靶点。新鲜的 G-CSF动员的外周血中成熟和未成熟DC细胞 将被感染，以预测使用慢病毒载体 体内免疫。特别是，调查人员将监测是否 转导功能障碍抗原提呈功能的发展 未成熟DC，以其产生HIV特异性CTL和 表达适当的共刺激分子和辅助分子。如果 必要时，调查人员会将其整合到病毒载体 细胞因子如Flt3配体(FL)或IL-4，以促进成熟 新鲜的华盛顿。具体目标3将验证体内转导是可行的 在小鼠模型中。研究人员将首先给小鼠接种 携带CMV启动子的HIV载体转导同基因DC及检测 脾和淋巴结中的病毒特异性CTL活性。用于概念证明 在体内转导方面，研究人员将在体内免疫小鼠 在每天注射FL后，载体的设计大大增加 体内DC前体细胞的数量。

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.