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

点击翻译按钮获取中文摘要

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.