Development of a broadly protective subunit vaccine against Pseudomonas aeruginosa

总结 铜绿假单胞菌是自然界中普遍存在的细菌，也是人类重要的机会致病菌 病原体，可导致烧伤、严重创伤、肺炎和正在接受治疗的患者严重感染 医院手术，尤其是需要插管或导管插入术的手术。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， 囊性纤维化（CF）患者肺部感染的原因，超过70%的CF患者被以下细菌定植 成年这些感染会降低肺功能，通常会降低患者的生活质量并导致早期死亡。 虽然已经有Pa疫苗在开发中，但没有一种疫苗获得许可。 与许多革兰氏阴性病原体一样，假单胞菌属（Pseudomonas spp.）具有III型分泌系统（T3 SS）， 能够避免宿主先天免疫的毒力因子，是感染开始所必需的。 在结构上类似于具有外部针的分子注射器，T3 SS装置（T3 SA）提供了一种分子注射器。 从细菌细胞质进入宿主细胞的能量管道。一种针尖蛋白质和两种蛋白质中的第一种 转运蛋白定位于T3 SA针的远端以介导宿主细胞接触。这些蛋白质， PcrV和PopB分别是假单胞菌致病所需的，并且在假单胞菌中95-98%保守。 假单胞菌相当保守的志贺氏菌T3 SA尖端和转运蛋白iPad和IpaB， 分别融合，得到DBF，我们已经表明，作为一种新的亚单位疫苗抗原保护小鼠 针对同源和异源志贺菌属的挑战。同样，高度保守的T3 SA尖端和 转运蛋白融合保护小鼠抵抗博德特氏菌属，肠道沙门氏菌和伯克霍尔德氏菌属。在 在初步的小鼠实验中，将PcrV/PopB融合物PaF与粘膜佐剂混合， 鼻内给药在这个最初的初步实验中，100%的保护（44%的灭菌免疫）， 对于PaF接种的小鼠可见。然而，60%的PBS对照小鼠也存活了下来（尽管没有 杀菌免疫）。尽管如此，在这个高风险，高回报的R21中，我们将在小鼠中优化疫苗 模型，并将研究结果扩展到公认的囊性纤维化大鼠模型。 因此，由于PaF+dmLT疫苗似乎保护小鼠免受Pa肺攻击， 44%杀菌免疫，PaF+dmLT将引发体液和细胞介导的免疫应答， 保护大多数Pa菌株，包括那些导致CF患者疾病的菌株。两个具体目标 本研究的主要内容是：1）评估由以下引起的相应的体液和细胞介导的免疫应答： PaF+dmLT和2）确定PaF+dmLT针对Pa的保护功效。

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.