A prophylactic vaccine to prevent colonization by Pseudomonas aeruginosa

项目摘要 疫苗接种是我们这个时代最伟大的公共卫生成就。随着抗生素耐药性的爆发， 针对多重耐药（MDR）细菌病原体的新疫苗比以往任何时候都更重要。 铜绿假单胞菌（Pa）是一种机会致病菌，可引起严重的感染 囊性纤维化（CF）、烧伤、严重创伤、肺炎以及需要插管的危重患者 或导管插入术。清除Pa已经成为问题，因为它已经变得越来越耐抗生素。这 与MDR Pa相关的负面结局的最大风险因素是晚期， 年龄60岁以后，MDR Pa导致的发病率和死亡率显著增加。虽然有 PA疫苗正在开发中，没有一种获得许可。 与许多革兰氏阴性病原体一样，PAO 1/PA 14进化枝的Pa菌株具有III型分泌物 系统（T3 SS），一种毒力因子，允许避免宿主先天免疫，并且是 感染T3 SS装置（T3 SA）的结构类似于带有外部针头的分子注射器， 提供了从细菌细胞质进入宿主细胞的能量通道，用于转运效应蛋白 介导感染的关键方面。针尖蛋白和两个转运蛋白中的第一个定位于 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 致病所需的，95-98%保守的Pa。我们已经融合了PcrV和PopB以给出PaF。 在证明了PaF的保护功效后，我们将LTA 1基因融合，LTA 1是不稳定毒素的活性部分， 从ETEC到PaF的N末端（L-PaF）。L-PaF显著降低小鼠和大鼠的肺负荷。当 与PBS接种的小鼠相比，L-PaF接种的小鼠具有显著更高的OPK活性， 从肺细胞分泌血清和升高水平的IL-17。最近，Pa异常值已经被确定， 完全缺乏T3 SS并使用ExlA破坏宿主细胞膜。因此，我们将ExlA添加到我们的 L-PaF（L-PaFE）乳剂，并已证明鼻内给药时对PAO 1/14/7分支具有保护作用。 此外，我们还添加了BECC 438，一种新型单磷酰脂质A（MPL）生物类似药（TLR 4激动剂）， 增加OPK活性（L-PaFEB 438）。R 01的目标是继续发展我们广泛的保护性 通过评估啮齿动物模型中的保护性免疫应答来确定PA疫苗制剂。知道 这些模型中的疫苗效力和免疫应答将使我们能够最终确定疫苗配方， 证明了该制剂在人类中的潜在效用。

PROJECT SUMMARY Vaccination is the greatest public health achievement of our time. With an explosion of antibiotic resistance, new vaccines against multi-drug resistant (MDR) bacterial pathogens are more important than ever. Pseudomonas aeruginosa (Pa) is an opportunistic human pathogen that causes severe infections in patients with cystic fibrosis (CF), burns, severe wounds, pneumonia, as well as critically ill patients who require intubation or catheterization. Clearing Pa has become problematic as it has become increasingly antibiotic resistant. This is exacerbated by the fact that the biggest risk factor for negative outcomes associated with MDR Pa is advanced age. After 60, there is a significant increase in morbidity and mortality resulting from MDR Pa. While there are Pa vaccines in development, none are licensed. Like many Gram-negative pathogens, Pa strains of the PAO1/PA14-clades 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 for transporting effector proteins that mediate key aspects of infection. 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 pathogenesis and are 95-98% conserved among Pa. We have fused PcrV and PopB to give PaF. After demonstrating the protective efficacy of PaF, we genetically fused LTA1, the active moiety of labile toxin from ETEC, to the N-terminus of PaF (L-PaF). L-PaF reduces mouse and rat lung burdens significantly. When compared to PBS-vaccinated mice, L-PaF-vaccinated mice possessed significantly higher OPK activity in the sera and elevated levels of IL-17 were secreted from lung cells. Recently, Pa outliers have been identified that are devoid of the T3SS entirely and use ExlA to disrupt host cell membranes. Thus, we have added ExlA to our L-PaF (L-PaFE) emulsion and have demonstrated protection in PAO1/14/7 clades when delivered intranasally. Furthermore, we have added BECC438, a novel monophosphoryl lipid A (MPL) biosimilar (a TLR4 agonist), to increase OPK activity (L-PaFEB438). The goal of the R01 is to continue to develop our broadly protective Pa vaccine formulation by assessing the protective immune response in rodent models. Knowing the vaccine efficacy and immune response in these models will allow us to finalize the vaccine formulation and the demonstrate the potential utility of that formulation in humans.