STIMULATION OF HIV SPECIFIC CTL WITH TOXIN FUSIONS

尽管过去研究的大多数艾滋病毒疫苗方法 专注于产生针对病毒的中和抗体， 越来越多的证据表明， 奋进在于引发细胞抗病毒免疫的策略。 申请人已经提出了旨在开发一种 具有引发细胞毒性T淋巴细胞能力的肽疫苗 (CTL)体内抗HIV-1表位的应答。 麻醉和呕吐 蛋白质已被确定为潜在重要的位点， 是他们工作的缩影，也将是他们工作的重点。 为了 为了引发CTL应答，含有CTL表位的蛋白质必须 递送至哺乳动物细胞的胞质溶胶。 在那里，它被处理成 形成肽，然后与MH I类分子复合 并呈现在细胞表面。 最近的研究揭示了 细胞内作用的毒素，例如炭疽毒素（AT），可以 被修饰以消除其毒性并使其能够递送异源的 蛋白质到哺乳动物细胞胞质溶胶。 作为这些的衍生物， 根据这些发现，申请人已经证明AT融合蛋白 含有选自单核细胞增生李斯特菌的表位， 淋巴细胞性脉络膜脑膜炎病毒可用于引发保护性 小鼠对这些病原体的CTL应答。 研究中 提出，申请人打算制备类似的AT融合物， 继续nef和gag蛋白并测试这些构建体的 在小鼠中引发抗原特异性CTL应答的能力。 此外，申请人还将试图确定是否 用这些融合体体外处理的人细胞可以诱导增殖 从例患者外周血中提取gag和nef特异性CTL 感染了艾滋病病毒 最后，研究人员将调查 患者来源的gag和nef特异性CTL可以裂解单倍型匹配的 用融合蛋白处理的靶细胞。 这些实验将 允许使用基于AT的疫苗策略来产生CTL 评估对HIV抗原的反应，如果呈阳性， 获得适应症，将为安全性和有效性奠定基础 基于AT的疫苗在灵长类动物模型中的研究。

Although most HIV vaccine approaches investigated in the past have focused on generating neutralizing antibodies against the virus, there is increasing evidence to suggest that the key to success of this endeavor lies in strategies for eliciting cellular antiviral immunity. The applicants have proposed experiments aimed at developing a peptide vaccine with the capacity to prime a cytotoxic T- lymphocyte (CTL) response in vivo aginst HIV-1 epitomes. The nef and gag proteins have been identified as the sites of potentially important epitomes and will be the primary focus of their work. In order to prime a CTL response, a CTL epitome-containing protein must be delivered to the cytosol of mammalian cells. There, it is processed to form peptides which are then complexed with MH class I molecules and presented at the cell surface. Recent studies have revealed how the intra cellularly acting toxin, such as the anthrax toxin (AT), may be modified to ablate its toxicity and enable it to deliver heterologous proteins to the mammalian cell cytosol. As a ramification of these findings, the applicants have demonstrated that AT fusion proteins containing selected epitomes from Listeria monocytogenes and lymphocytic choriomenigitis virus may be used to prime protective CTL responses in mice against these pathogens. In the research proposed, the applicants intend to prepare similar AT fusions continuing the nef and gag proteins and test these constructs for ability to prime an antigen-specific CTL response in mice. Furthermore, the applicants will also attempt to determine whether human cells treated invitro with these fusions can induce proliferation of gag- and nef-specific CTL from peripheral blood ofpatients infected with HIV. Finally, the researchers will investigate whether patient-derived gag- and nef-specific CTL can lyse haplotype-matched target cells treated with the fusion proteins. These experiments will allow the utility of the AT-based vaccine strategy to generate a CTL response against HIV antigens to be evaluated and, if positive indications are obtained, will set the stage for safety and efficacy studies of AT-based vaccines in primate animal models.