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，自然地将这种生物体的减毒衍生物靶向这些诱导位点，并允许在开发粘膜HIV- 1 DNA疫苗中利用这一特性。因此，我们和其他人最近报道了基因工程，减毒沙门氏菌菌株能够将DNA疫苗递送到小鼠组织，在那里它们引发针对DNA疫苗编码的乘客抗原的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CD8+ T细胞数量与单杯注射的裸Env DNA疫苗诱导的数量相似。该项目的主要目标是获得动物模型中由减毒沙门氏菌递送的HIV-1 Env DNA疫苗的安全性和免疫原性的临床前数据。项目3需要这些数据来验证我们的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。我们将获得这些数据的具体目的是：1)构建和表征一种原型减毒伤寒沙门菌HIV-1 gp120 DNA疫苗载体：我们将构建两种HIV-1/Ba-L gp120 DNA疫苗，并将其引入我们的减毒伤寒沙门菌载体菌株CVD DELTA/ASD。第一种将包括HIV-1-Ba-L分离物的gp120 DNA疫苗（gp120Ba-L）。该gp120已经过密码子优化，可在哺乳动物细胞中表达。第二种将被编码gp120-CD4融合蛋白的嵌合基因破坏，该蛋白有望为广泛中和抗HIV-11的抗体做准备（参见项目2）；2)获得目的1中开发的HIV-1沙门氏菌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疫苗载体构建的临床前安全性、免疫原性和遗传毒性数据。虽然斑疹伤寒沙门氏菌仅对人类具有完全毒力，但已经建立了小鼠模型来评估活淋巴样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，以在项目3中描述的志愿者中进行1期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.