Development of a broad spectrum glycoconjugate vaccine to prevent wound and disseminated infections with Klebsiella pneumoniae and Pseudomonas aeruginosa.

Klebsiella pneumoniae (KP) and Pseudomonas aeruginosa (PA) are important human pathogens that are associated with a range of infection types, including wound and disseminated infections. Treatment has been complicated by rising rates of antimicrobial resistance. Immunoprophylactic strategies are not constrained by antimicrobial resistance mechanisms. Vaccines against these organisms would be important public health tools, yet they are not available. KP surface O polysaccharides (OPS) are protective antigens in animal models of infection. Similarly, PA flagellin (Fla), the major subunit of the flagellar filament, is required for virulence and is a target of protective antibodies in animal models. We report herein the development of a combined KP and PA glycoconjugate vaccine comprised of the four most common KP OPS types associated with human infections (O1, O2, O3, O5), chemically linked to the two Fla types of PA (FlaA, FlaB). Conjugation of KP OPS to PA Fla enhanced anti-polysaccharide immune responses and produced a formulation that generated antibody titers to the four KP OPS types and both PA Fla antigens in rabbits. Passive transfer of vaccine-induced rabbit antisera reduced the bacterial burden and protected mice against fatal intravenous KP infection. Mice passively transferred with conjugate-induced antisera were also protected against PA infection after thermal injury with a FlaB-expressing isolate, but not a FlaA isolate. Taken together, these promising preclinical results provide important proof-of-concept for a broad spectrum human vaccine to prevent KP and PA infections.

High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication.

Pseudomonas aeruginosa as an opportunistic pathogen is a significant cause of acute and chronic infections in patients with compromised defenses. This bacterium is motile via a single polar flagellum made of polymerized flagellin subunits differentiated into two major serotypes: A and B. flagellin plays an important role as a virulence factor in the adhesion, colonization and invasion of P. aeruginosa into host epithelial cells. To develop a functional vaccine that can be used in practical application to prevent and treat infection, type B-flagellin was produced as recombinant protein. In this work, the fliC gene was introduced into a pET28a vector and expressed in Escherichia coli BL21 (DE3). The expressed recombinant protein was purified by a modified method without sonication using a HisTrap affinity column. The functional activities of produced flagellin were confirmed by ELISA, western blot analysis, motility inhibition assay and opsonophagocytosis test. The purification process of the type B-flagellin was lead to a high yield. The produced recombinant type B-flagellin showed high biological activity in all of these standard assays. In conclusions, this report provides the new protocol to efficiently obtain the type B-flagellin with high biological activity and immunogenicity. This immunogen can be introduced as an adjuvant or vaccine in the future study.

Protective efficacy of Pseudomonas aeruginosa type-A flagellin in the murine burn wound model of infection.

The main goal of this study was to develop a vaccination strategy that would enhance the protective response against the recombinant type A flagellin (r-fla-A) of Pseudomonas aeruginosa in the burn wound sepsis model. Inbred mice were immunized with r-fla-A with or without alum adjuvant. The vaccinated mice were burned and challenged with P. aeruginosa. To evaluate the type of induced immune response, sera were analyzed by ELISA for total IgG, IgG1, and IgG2a isotypes. To determine the functional activity of anti r-fla IgG, opsonophagocytic killing and motility inhibition assay was performed. In vivo administration of r-fla-A afforded a remarkable improvement in survival of mice (83.3%) challenged with homologous strain (PAK) in the burn wound infection. The antibodies generated against the r-fla-A achieved 25% survival in immunized mice that had been infected with heterologous strain PAO1. Flagellin also induced high level humoral immune response via high titers of serum IgG1 in the burn and challenged mice. Anti r-fla-A antibody promoted phagocytosis of the PAK strain, and the number of viable bacterial cells decreased over 53.1%; In contrast, low opsonophagocytic killing activity (17.4%) was observed when the antiserum to r-fla-A was treated with the PAO1 strain. The anti r-fla-A antisera was able to inhibit the motility of the homologous strains; however, they did not inhibit the heterologous strains. We concluded that active immunization with recombinant type A-flagellin could protect burn mice against lethal P. aeruginosa challenge via immobilization of the pathogen which promoted the phagocytic activity.

Pseudomonas immunotherapy: a historical overview.

The historic development of vaccines to be used as immunotherapy for Pseudomonas aeruginosa infections, in various patient populations, is reviewed. Commentary is offered concerning the relevance of each approach in light of our current understanding of the pathological process of these infections.

Assessment of a silver-coated barrier dressing for potential use with skin grafts on excised burns.

Acticoat burn dressing is a silver-coated dressing with antimicrobial activity purported to reduce infection from environmental organisms in partial and full-thickness wounds. Acticoat was tested for activity as an antimicrobial treatment and as an antimicrobial barrier dressing in three in vitro assays. It was found that a modified disc assay method gave false negative results but in an assay in which bacteria were inoculated on top of samples of Acticoat, bacterial numbers were reduced, over time, with all microorganisms tested. Acticoat served as a barrier for bacteria, inoculated onto it, from contaminating the surface of an agar plate under the Acticoat. The data show that Acticoat has: antimicrobial capabilities, but to be effective hours of contact between Acticoat and the microorganisms are required; and the capacity to serve as an antimicrobial barrier dressing. These findings support the conclusion that Acticoat has activity to reduce microbial contamination of wounds from environmental sources.

Effect of antibiotics on polymorphonuclear neutrophil apoptosis.

STUDY OBJECTIVE: To evaluate the effects of various antibiotics-direct and indirect as a result of bacterial killing-on polymorphonuclear neutrophil (PMN) apoptosis. DESIGN: In vitro analysis. SETTING: Research laboratory. INTERVENTION: Whole blood collected from healthy human subjects was incubated with and without Klebsiella pneumoniae (1.0 x 10(5) colony-forming units [cfu]/ml) plus ceftazidime 50 microg/ml, gentamicin, ciprofloxacin, trovafloxacin, tetracycline, doxycycline, erythromycin, azithromycin (each 5 microg/ml), or lipopolysaccharide 10 microg/ml. After staining with fluorescein-conjugated annexin V, red blood cells were lysed, and the remaining white blood cells were assessed by flow cytometry with gating on PMNs. MEASUREMENTS AND MAIN RESULTS: In the absence of K. pneumoniae infection, antibiotic exposure directly decreased PMN apoptosis by 17.8% (range -25.0% to -13.9%, p=0.008) compared with untreated cells. In the presence of K. pneumoniae, all antibiotic treatments, even those with poor in vitro activity for the bacterial isolate, decreased PMN apoptosis by 26.2% (range -38.0% to -17.8%, p<0.001) compared with untreated control cells and by 36.6% compared with untreated (no antibiotic) K. pneumoniae-stimulated cells (range -46.2% to -28.0%, p<0.001). CONCLUSIONS: All tested antibiotics in clinically relevant concentrations resulted in modest yet consistent decreases in PMN apoptosis. The magnitude of this change increased slightly in the presence of K. pneumoniae infection. In vivo studies are needed to determine whether antibiotic-associated prolongation of PMN survival improves host response to infection.

Secreted products of a nonmucoid Pseudomonas aeruginosa strain induce two modes of macrophage killing: external-ATP-dependent, P2Z-receptor-mediated necrosis and ATP-independent, caspase-mediated apoptosis.

A nonmucoid clinical isolate of Pseudomonas aeruginosa, strain 808, elaborated ATP-dependent and ATP-independent types of cytotoxic factors in the growth medium. These cytotoxic factors, active against macrophages, were secreted during the exponential phase of growth in a complex medium. Commensurate with the appearance of the cytotoxic activities in the cell-free growth medium, several ATP-utilizing enzymic activities, such as adenylate kinase, nucleoside diphosphate kinase and 5'-nucleotidase (ATPase and/or phosphatase), were detected in the medium. These ATP-utilizing enzymes are believed to convert external ATP, presumably effluxed from macrophages, to various adenine nucleotides, which then activate purinergic receptors such as P2Z, leading to enhanced macrophage cell death. Pretreatment of macrophages with periodate-oxidized ATP (oATP), which is an irreversible inhibitor of P2Z receptor activation, prevented subsequent ATP-induced macrophage cell death. A second type of cytotoxic factor(s) operated in an ATP-independent manner such that it triggered activation of apoptotic processes in macrophages, leading to proteolytic conversion of procaspase-3 to active caspase-3. This cytotoxic factor(s) did not appear to act on procaspase-3 present in macrophage cytosolic extracts. Intact macrophages, when exposed to the cytotoxic factor(s) for 6-16 h, underwent apoptosis and demonstrated the presence of active caspase-3 in their cytosolic extracts. Interestingly, two redox proteins, azurin and cytochrome c(551), were detected in the cytotoxic preparation. When cell-line-derived or peritoneal macrophages or mast cells were incubated overnight with Q-Sepharose column flow-through fraction or with a mixture of azurin and cytochrome c(551), they underwent extensive cell death due to induction of apoptosis.