An intranasal room stable vaccine formulation to prevent Pseudomonas aeruginosa (R21AI169691)

项目摘要 疫苗接种也许是我们这个时代最伟大的公共卫生成就。随着抗生素的激增 耐药性，开发针对多药耐药（MDR）细菌病原体的新疫苗， 比以往更重要。铜绿假单胞菌（Pseudomonasaeruginosa，Pa）是一种重要的人类机会致病菌， 导致囊性纤维化（CF）、烧伤、严重创伤、肺炎以及 需要插管或导管插入术的危重患者。清除Pa已成为一个问题， 抗生素耐药性越来越强。这是加剧了这一事实，即最大的风险因素，为消极的 与MDR Pa相关的结局是高龄。60岁以后， MDR Pa导致的发病率和死亡率。虽然有Pa疫苗正在开发中，但没有一种是 有执照。R21的目标是定义用于我们的预防性治疗的稳定的纳米颗粒（NP）悬浮液。 在建立生物膜之前预防Pa的Pa疫苗，无论菌株如何。 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 T3 SS是一种能够避免宿主先天免疫的系统，是启动感染所必需的。 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。 从细菌进入宿主细胞的能量管道，用于转运介导关键蛋白的效应蛋白。 感染的方面。针尖蛋白和两个转运蛋白中的第一个定位于 T3 SA针介导宿主细胞接触。在Pa中，这些蛋白质分别是PcrV和PopB，并且它们 是发病所必需的。它们在所有铜绿假单胞菌菌株中也是高度保守的（95-98%）， 拥有T3 SS。我们已经融合了PcrV和PopB以给出PaF。促进同时摄取抗原 和佐剂的抗原呈递细胞，我们基因融合LTA 1，活性部分的dmLT，以N- PaF末端（L-PaF）。当受到攻击时，L-PaF显着降低小鼠和大鼠肺Pa负担 PAO 1/PA 14进化枝Pa.最近，PAO 7进化枝的Pa离群值已经被鉴定为缺乏 T3 SS，而是使用胞外溶素A（ExlA）来破坏宿主细胞膜。我们在L-PaF中添加了ExlA 经鼻内给药时，已证明可保护患者免受PAO 1/14和PAO 7的侵害。 小鼠中的进化枝。来自这些小鼠的血清表现出显著的调理吞噬杀伤（OPK）。此外，高 从L-PaF接种的小鼠的肺细胞分泌IL-17水平。IL-17和OPK都被认为是 对清除Pa感染很重要。 在该R21中，我们将评估稳定的颗粒ExlA/L-PaF NP的保护性免疫应答。 悬浮液我们将在两个目标完成这个项目：1）我们将产生纳米颗粒制剂的 ExlA/L-PaF抗原并评估其稳定性。2)然后我们将确定由以下物质引起的免疫应答： ExlA/L-PaF纳米颗粒制剂，并测定其从肺中清除Pa的能力。

PROJECT SUMMARY Vaccination is perhaps the greatest public health achievement of our time. With an explosion of antibiotic resistance, developing new vaccines against multi-drug resistant (MDR) bacterial pathogens is more important than ever. Pseudomonas aeruginosa (Pa) is an important 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 the age of 60, there is a significant increase in morbidity and mortality resulting from MDR Pa. While there are Pa vaccines in development, none are licensed. The goal of the R21 is to define a stable nanoparticle (NP) suspension for our prophylactic Pa vaccine that prevents Pa, regardless of strain, prior to establishment of a biofilm. Like many Gram-negative pathogens, Pa strains of the PAO1/PA14-clades possess a type III secretion system (T3SS) that allows avoidance of host innate immunity and is required for initiating infection. Structurally resembling a molecular syringe with an external needle, the T3SS apparatus (T3SA) provides an energized conduit from the bacterium into the host cell for transporting the 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. In Pa these proteins are PcrV and PopB, respectively, and they are required for pathogenesis. They are also highly conserved (95-98%) among all P. aeruginosa strains that possess a T3SS. We have fused PcrV and PopB to give PaF. To promote simultaneous uptake of antigen and adjuvant by antigen presenting cells, we genetically fused LTA1, the active moiety of dmLT, to the N- terminus of PaF (L-PaF). L-PaF reduces mouse and rat lung Pa burden significantly when challenged with a PAO1/PA14 clade Pa. Recently, Pa outliers of the PAO7 clade have been identified that are devoid of the T3SS and instead use exolysin A (ExlA) to disrupt host cell membranes. We have added ExlA to our L-PaF formulation and, when delivered intranasally, have demonstrated protection against PAO1/14 and PAO7 clades in mice. Sera from these mice exhibit significant opsonophagocytic killing (OPK). Additionally, elevated levels of IL-17 were secreted from lung cells of L-PaF-vaccinated mice. Both IL-17 and OPK are deemed important in clearing Pa infections. In this R21, we will assess the protective immune response of a stable particulate ExlA/L-PaF NP suspension. We will complete this project in two aims: 1) We will generate nanoparticle formulations for the ExlA/L-PaF antigens and assess their stability. 2) We will then determine the immune response(s) elicited by the ExlA/L-PaF nanoparticle formulation(s) and determine their ability to clear Pa from the lungs.