Evaluation of Protein Channel-Attenuated Salmonella Vaccines

描述（由申请人提供）：该项目的长期目标包括通过使用沙门氏菌中的定植因子抗原I （CFA/I）获得的异源基因介导灭活来开发活疫苗。我们怀疑这种衰减是由于通道CfaC的过表达，而不是由于纤维CfaBE，尽管CfaBE可以增强通道介导的衰减。由于这种衰减策略以前没有描述过，我们质疑它是否可以用于沙门氏菌疫苗的开发。由于传统的基于基因失活的活疫苗开发方法遇到了障碍，本研究将开发用于人类的沙门氏菌活疫苗。因此，我们设定了以下两个特定目的：特定目的1：研究是否毛状亚基的缺失，分泌装置的缺失，或者两者都是导致野生型沙门氏菌衰减的原因。特异性目标2：确定cfa/I操纵子的哪一种缺失突变体可以减弱野生型沙门氏菌，从而可以用作沙门氏菌病的保护性疫苗。为了实现这两个目标，我们将在沙门氏菌细胞外膜（OM）中安装CfaC通道。通道沙门氏菌将通过原子力显微镜（AFM）评估细胞扩展，在各种条件下的生存能力，通过场发射扫描电子显微镜（FESEM）评估形态学，通过巨噬细胞评估体外毒力，通过小鼠模型评估体内毒力。由于细胞OM中的通道CFA/I允许741道尔顿红霉素进入，它也允许其他较小的分子，如水、离子和有毒分子通过。因此，细胞OM渗透平衡将部分失去，这将导致细菌病原体的衰减。这项研究将有可能开发出新的人用沙门氏菌活疫苗。公共卫生相关性：拟议的项目题为“蛋白质通道减毒沙门氏菌疫苗的评价”，将构建一种旨在预防人类沙门氏菌病的活疫苗。肠炎沙门氏菌是一种引起多种食物和水传播疾病的疾病，如肠胃炎和伤寒。它可以感染广泛的动物宿主和人类。在三种常见的伤寒疾病中，据估计，仅伤寒沙门氏菌就造成了大约1600万例病例，全世界每年有60万人死亡，最近在美国每年有600人死亡。使情况更糟的是，这种疾病对以前用于治疗它的抗生素产生了耐药性。最近，我们实验室发明了一种灭活沙门氏菌的新方法。这种新策略与传统的毒力基因失活方法完全不同。如果证明对沙门氏菌有效，这种策略也将适用于其他肠杆菌。因此，该项目不仅有利于美国，也有利于其他国家沙门氏菌病的流行。

DESCRIPTION (provided by applicant): The long-term objective of the project involves developing live vaccines through heterologous gene-mediated inactivation, as obtained using colonization factor antigen I (CFA/I) in Salmonella. We suspect this attenuation is because of the overexpression of channel CfaC and not due to the fimbriae CfaBE, although CfaBE may enhance the channel-mediated attenuation. Since this attenuation strategy was not previously described, we questioned whether it could be utilized for Salmonella vaccine development. Since conventional live vaccine development methods based on gene inactivation have been met with obstacles, this proposed study will generate Salmonella live vaccines for application in humans. As such, we set the following two Specific Aims: Specific Aim 1: Examine whether the deletion of fimbrial subunits, the secretion apparatus, or both are responsible for the observed attenuation of wild-type Salmonella. Specific Aim 2: Determine which of the deletion mutants of the cfa/I operon attenuates wild-type Salmonella and can therefore be used as a protective vaccine against salmonellosis. To achieve these two aims, we will install CfaC channel in the Salmonella cell outer membrane (OM). The channeled Salmonella will be evaluated for cell extension via atomic force microscope (AFM), viability in various conditions, morphology via Field Emission Scanning Electron Microscopy (FESEM), in vitro virulence via macrophages, and in vivo virulence by mouse model. Since the channel CFA/I in cell OM allows 741 dalton erythromycin to enter, it will also permit other smaller molecules, such as water, ions, and toxic molecules to pass. Thus, cell OM osmotic balance will be partially lost, which will result in the attenuation of bacterial pathogens. This study will possibly develop new live vaccines against Salmonella for human usage. PUBLIC HEALTH RELEVANCE: The proposed project entitled, "Evaluation of Protein Channel-Attenuated Salmonella Vaccines" will construct a live vaccine that aims at preventing human salmonellosis. Salmonella enterica is a disease that causes a variety of food and water-borne illnesses, such as gastroenteritis and typhoid fever. It can infect a wide range of animal hosts and also humans. Of the three common Typhoid fever disease, S. Typhi alone is estimated to cause approximately 16 million cases, with 600,000 deaths annually worldwide and 600 fatalities recently occurring each year in the United States. To make the situation even worse, this disease has acquired resistance to the antibiotics previously used to treat it. Recently, our laboratory invented a new strategy for inactivating the Salmonella. This new strategy is totally different from the conventionally used virulence gene inactivation method. If demonstrated effective in Salmonella, this strategy will be also applicable to other enterobacteria, as well. Therefore, this project will benefit not only the USA but also other countries epidemic with salmonellosis.