SALMONELLA GP120 DNA VACCINE VECTOR

沙门氏菌对肠粘膜表面淋巴组织中的树突状细胞和巨噬细胞的趋向性，自然将这种生物的减毒衍生物靶向这些诱导部位，并允许在开发粘膜HIV-1DNA疫苗时利用这一特性。因此，我们和其他人最近报告了经过基因工程的减毒沙门氏菌菌株能够将DNA疫苗运送到小鼠组织，在那里它们针对DNA疫苗编码的乘客抗原引发体液和细胞介导的免疫反应。我们实验室产生的初步数据还表明，单剂量口服沙门氏菌HIGV-1Env DNA疫苗诱导的干扰素-γ分泌CD8T细胞数量与肌肉注射单次MUG剂量的裸露Env DNA疫苗诱导的数量相似。本项目的主要目的是在动物模型中获得由减毒沙门氏菌提供的HIV-1Env DNA疫苗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计划的主要假设，即这种疫苗在人类志愿者中是安全和免疫原性的。1)构建和鉴定减毒伤寒沙门氏菌gp120DNA疫苗载体：我们构建了两个HIV-1/巴-L gp120DNA疫苗，并将其导入我们的减毒伤寒沙门氏菌载体株CVD Delta/asd。第一个将包括艾滋病毒-1-巴-L分离株的gp120DNA疫苗(gp120巴-L)。该gp120已经过密码子优化，可以在哺乳动物细胞中表达。第二项研究将涉及编码gp120-CD4融合蛋白的嵌合基因，该融合蛋白有望用于广泛中和抗HIV-11抗体(另见项目2)；以及2)获得在AIM 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，我们将获得关于沙门氏菌HIV-1 DNA疫苗载体构建的临床前安全性、免疫原性和遗传毒性数据。虽然伤寒沙门氏菌只对人类具有完全的毒力，但已经开发了小鼠模型来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应用，如项目3所述，在志愿者中进行第一阶段安全性和免疫原性研究。

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.