SALMONELLA GP120 DNA VACCINE VECTOR

沙门氏菌对肠粘膜表面的淋巴组织中的树突状细胞和巨噬细胞的嗜性天然地将该生物体的减毒衍生物靶向这些诱导位点，并允许在粘膜HIV- 1 DNA疫苗的开发中利用该特性。因此，我们和其他人最近报道了基因工程的减毒沙门氏菌菌株能够将DNA疫苗递送至小鼠组织，在那里它们引发针对由DNA疫苗编码的乘客抗原的体液和细胞介导的免疫应答。我们实验室产生的初步数据也表明，单次口服剂量的沙门氏菌HIGV-1 Env DNA疫苗诱导的分泌干扰素-γ的CD 8 + T细胞的数量与肌肉注射单杯剂量的裸Env DNA疫苗诱导的数量相似。本项目的主要目的是获得减毒沙门氏菌递送的HIV-1 Env DNA疫苗在动物模型中的安全性和免疫原性的临床前数据。项目3需要这些数据来检验我们的IPCAVD项目的主要假设，即这些疫苗在人类志愿者中是安全的和具有免疫原性的。1）构建和鉴定减毒伤寒沙门氏菌HIV-1 gp 120 DNA疫苗载体：我们将构建两种HIV-1/Ba-L gp 120 DNA疫苗，并将它们导入我们的减毒伤寒沙门氏菌载体株CVD DELTA/ASD中。第一个将由HIV-1-Ba-L分离株的gp 120 DNA疫苗（gp 120 Ba-L）组成。该gp 120已针对哺乳动物细胞中的表达进行了密码子优化。第二个将是编码gp 120-CD 4融合蛋白的嵌合基因的妥协，该融合蛋白预计将引发针对HIV-11的广泛中和抗体（另见项目2）;以及2）获得关于目标1中开发的沙门氏菌HIV-1 DNA疫苗的临床前安全性、免疫原性和遗传毒性数据。在本研究的第二阶段，我们将获得沙门氏菌HIV-1 DNA疫苗载体构建体的临床前安全性、免疫原性和遗传毒性数据。尽管伤寒沙门氏菌仅对人类具有完全毒性，但已开发出小鼠模型用于评估活淋巴疫苗的安全性和免疫原性。从这些研究中获得的信息将用于IND申请，以在志愿者中进行I期安全性和免疫原性研究，如项目3所述。

The tropism of Salmonella for dendritic cells and macrophages in the lymphoid tissue of the intestinal mucosal surface, naturally targets attenuated derivatives of this organism to these inductive sites and allows for the exploitation of this property in the development of a mucosal HIV- 1 DNA vaccine. Accordingly, we and others have recently reported that genetically-engineered, attenuated Salmonella strains are capable of delivering DNA vaccines to mouse tissues where they elicit humoral and cell-mediated immune responses against passenger antigens encoded by the DNA vaccine. Preliminary data generated in our laboratory also suggests that a single oral dose of a Salmonella HIGV-1 Env DNA vaccine induces similar numbers of interferon-gamma-secreting CD8+ T cells as those induced by the naked Env DNA vaccine injected intramuscularly as a single mug dose. The principle objective of this project is to obtain preclinical data on the safety and immunogenicity of HIV-1 Env DNA vaccines delivered by attenuated Salmonella in animal models. This data is required by Project 3 to test the principal hypothesis on our IPCAVD program that such vaccines are safe and immunogenic in human volunteers. We will obtain this data in the following specific aims: 1) to construct and characterize a prototype attenuated Salmonella typhi HIV-1 gp120 DNA vaccine vector: We will construct two HIV-1/Ba-L gp120 DNA vaccines and introduce them into our attenuated Salmonella typhi vector strain, CVD DELTA/ASD. The first will be comprised of a gp120 DNA vaccine of the HIV-1-Ba-L isolate (gp120Ba-L). This gp120 has been codon-optimized for expression in mammalian cells. The second will be compromised of a chimeric gene encoding a gp120-CD4 fusion protein that is expected to prime for broadly neutralizing antibodies against HIV-11 (see also Project 2); and 2) to obtain preclinical safety, immunogenicity and genetic toxicity data on the Salmonella HIV-1 DNA vaccines developed in aim 1. In the second phase of this study we will obtain preclinical safety, immunogenicity and genetic toxicity data on the Salmonella HIV-1 DNA vaccine vector constructs. Although Salmonella typhi is only fully virulent in humans, murine models have been developed for the assessment live lymphoid vaccine safety and immunogenicity. The information gained from these studies will be used in IND applications to conduct Phase 1 safety and immunogenicity studies in volunteers as described in Project 3.