Development of a broadly protective subunit vaccine against Pseudomonas aeruginosa

摘要 铜绿假单胞菌是自然界中普遍存在的一种细菌，是人类重要的机会主义者 可在烧伤、严重创伤、肺炎和正在接受治疗的患者中引起严重感染的病原体 院内操作，尤指需要插管或导尿管的操作。清除铜绿假单胞菌(PA) 随着抗生素耐药性的增加，变得越来越困难。特别值得关注的是，爸爸是少校 囊性纤维化患者肺部感染的原因超过70%的囊性纤维化患者被 成人期。这些感染会降低肺功能，这往往会降低患者的生活质量，并导致过早死亡。 虽然已经有几种巴氏杆菌疫苗在开发中，但还没有一种获得许可。 像许多革兰氏阴性病原体一样，假单胞菌属(Pusomonas spp.)拥有III型分泌系统(T3SS)，a 一种毒力因子，可避免宿主的先天免疫，是感染开始时所必需的。 T3SS装置(T3SA)在结构上类似于带有外针的分子注射器，提供了一种 从细菌细胞质到宿主细胞的通电管道。一种针尖蛋白和两种蛋白质中的第一种 转运蛋白定位于T3SA针的远端，介导宿主细胞的接触。这些蛋白质， PcrV和POPB分别是假单胞菌致病所必需的，并且在 假单胞菌属同样保守的志贺氏菌T3SA末端和转位蛋白iPad和IPAB， 分别融合得到DBF，我们已经证明DBF是一种保护小鼠的新型亚单位疫苗抗原 抵抗同源和异源志贺氏菌的挑战。同样，高度保守的T3SA尖端和 转运子融合已经保护小鼠免受波尔德氏菌、肠沙门氏菌和伯克霍尔德氏菌的侵袭。在……里面 一项初步的小鼠实验，PAF，PcrV/POPB融合，与一种粘膜佐剂混合，并 鼻腔给药。在这个初步的初步实验中，100%的保护(44%的灭菌免疫力)是 以接种PAF疫苗的小鼠为例。然而，60%的PBS对照组小鼠也存活了下来(尽管没有 消毒免疫力)。尽管如此，在这种高风险、高回报的R21中，我们将在小鼠身上进行疫苗优化 并将这一发现推广到公认的囊性纤维化大鼠模型上。 因此，由于PAF+dmLT疫苗似乎能保护小鼠免受PA肺部攻击，因此 44%的杀菌免疫，PAF+dmLT将引发体液和细胞免疫反应， 保护大多数PA菌株，包括那些在CF患者中导致疾病的菌株。两个具体目标 这项研究的主要内容是：1)评估各自的体液和细胞介导的免疫反应 PAF+dmLT和2)PAF+dmLT对PA的保护作用。

SUMMARY Pseudomonas aeruginosa is a ubiquitous bacterium in nature and an important opportunistic human pathogen that can cause severe infections in patients with burns, severe wounds, pneumonia, and undergoing nosocomial procedures, especially those requiring intubation or catheterization. Clearing P. aeruginosa (Pa) has become progressively more difficult with growing antibiotic resistance. Of particular concern, Pa is the major cause of pulmonary infections in cystic fibrosis (CF) patients with over 70% of CF patients being colonized by adulthood. These infections decrease lung function that often reduce patient quality of life and cause early death. While there have been of Pa vaccines in development, none have been licensed. Like many Gram-negative pathogens, Pseudomonas spp. possess a type III secretion system (T3SS), a virulence factor that allows avoidance of host innate immunity and is required for the onset of infection. Structurally resembling a molecular syringe with an external needle, the T3SS apparatus (T3SA) provides an energized conduit from the bacterial cytoplasm into the host cell. A needle tip protein and the first of two translocator proteins localize to the distal end of the T3SA needle to mediate host cell contact. These proteins, PcrV and PopB, respectively, are required for Pseudomonas pathogenesis and are 95-98% conserved among Pseudomonas spp. The equivalent conserved Shigella T3SA tip and translocator proteins, IpaD and IpaB, respectively, were fused to give DBF, which we have shown as a novel subunit vaccine antigen protecting mice against challenges by homologous and heterologous Shigella spp. Similarly, highly conserved T3SA tip and translocator fusions have protected mice against Bordetella spp., Salmonella enterica and Burkholderia spp. In a preliminary mouse experiment, PaF, the PcrV/PopB fusion, was admixed with a mucosal adjuvant and delivered intranasally. In this initial preliminary experiment, 100% protection (with 44% sterilizing immunity) was seen for the PaF vaccinated mice. However, 60% of the PBS control mice also survived (although with no sterilizing immunity). Nevertheless, in this high risk, high reward R21, we will optimize the vaccine in the mouse model and to extend the findings to the accepted rat model of cystic fibrosis. Thus, because the PaF+dmLT vaccine appears to protect mice against Pa pulmonary challenge with 44% sterilizing immunity, PaF+dmLT will elicit a humoral and cell-mediated immune response that protects against most Pa strains, including those that cause disease in CF patients. The two specific aims of this investigation are: 1) Assess the respective humoral and cell-mediated immune responses elicited by PaF+dmLT and 2) Determine the protective efficacy of PaF+dmLT against Pa.