A novel anti-PcrV antibody providing enhanced protection against Pseudomonas aeruginosa in multiple animal infection models.

Pseudomonas aeruginosa is a major cause of hospital-acquired infections, particularly in mechanically ventilated patients, and it is the leading cause of death in cystic fibrosis patients. A key virulence factor associated with disease severity is the P. aeruginosa type III secretion system (T3SS), which injects bacterial toxins directly into the cytoplasm of host cells. The PcrV protein, located at the tip of the T3SS injectisome complex, is required for T3SS function and is a well-validated target in animal models of immunoprophylactic strategies targeting P. aeruginosa. In an effort to identify a highly potent and protective monoclonal antibody (MAb) that inhibits the T3SS, we generated and characterized a panel of novel anti-PcrV MAbs. Interestingly, some MAbs exhibiting potent inhibition of T3SS in vitro failed to provide protection in a mouse model of P. aeruginosa infection, suggesting that effective in vivo inhibition of T3SS with anti-PcrV MAbs is epitope dependent. V2L2MD, while not the most potent MAb as assessed by in vitro cytotoxicity inhibition assays, provided strong prophylactic protection in several murine infection models and a postinfection therapeutic model. V2L2MD mediated significantly (P < 0.0001) better in vivo protection than that provided by a comparator antibody, MAb166, a well-characterized anti-PcrV MAb and the progenitor of a clinical candidate, KB001-A. The results described here support further development of a V2L2MD-containing immunotherapeutic and may suggest even greater potential than was previously recognized for the prevention and treatment of P. aeruginosa infections in high-risk populations.

Effects of low-level inhalation exposure to cyclosarin on learned behaviors in Sprague-Dawley rats.

Behavioral and biochemical effects of low-level whole-body inhalation exposure to the chemical warfare nerve agent cyclosarin (GF) were evaluated. Sprague-Dawley rats were first trained on a variable-interval, 56-s (VI56) schedule of food reinforcement. The VI56 schedule specifies that a single lever press, following an average interval of 56 s, produces food reinforcement (i.e., a single food pellet). Subjects were then exposed to GF vapor at concentrations of 1.6-5.2 mg/m3, or air control, for 60 min. Following exposures, performance on the VI56 and acquisition and maintenance of a radial-arm maze (RAM) spatial memory task were evaluated during 55 test sessions over approximately 11 wk. GF exposures produced miosis in all subjects and other mild clinical signs of toxicity at the highest concentration. Convulsions were not observed in any subjects. GF exposures produced concentration-dependent decreases in acetylcholinesterase and butyrylcholinesterase activity. Additionally, blood assays revealed concentration-dependent levels of regenerated GF, thus verifying systemic exposure. The largest concentration of GF disrupted performance on the VI56 task. The deficit, however, resolved by the third postexposure test session. All subjects acquired, and maintained, performance on the RAM task, and no significant differences were seen as a result of GF exposure. No delayed effects from exposures were observed. These results demonstrate that, in rats, inhalation exposure to GF at levels below those producing convulsions and other severe clinical signs of toxicity may produce performance deficits on learned behaviors, but the deficits appear to not be persistent.

Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening.

Pseudomonas aeruginosa is a leading cause of hospital-associated infections in the seriously ill, and the primary agent of chronic lung infections in cystic fibrosis patients. A major obstacle to effective control of P. aeruginosa infections is its intrinsic resistance to most antibiotic classes, which results from chromosomally encoded drug-efflux systems and multiple acquired resistance mechanisms selected by years of aggressive antibiotic therapy. These factors demand new strategies and drugs to prevent and treat P. aeruginosa infections. Herein, we describe a monoclonal antibody (mAb) selection strategy on whole P. aeruginosa cells using single-chain variable fragment phage libraries derived from healthy individuals and patients convalescing from P. aeruginosa infections. This approach enabled identification of mAbs that bind three distinct epitopes on the product of the Psl. This exopolysaccharide is important for P. aeruginosa attachment to mammalian cells, and for the formation and maintenance of biofilms produced by nonmucoid and mucoid P. aeruginosa isolates. Functional screens revealed that mAbs to one epitope exhibit superior activity in opsonophagocytic killing and cell attachment assays, and confer significant protection in multiple animal models. Our results indicate that Psl is an accessible serotype-independent surface feature and promising novel protective antigen for preventing P. aeruginosa infections. Furthermore, our mAb discovery strategy holds promise for application to other bacterial pathogens.