Generation and Characterization of a Protective Monoclonal Antibody toPseudomonas aeruginosaPcrV

Abstract

Pseudomonas aeruginosa is a gram-negative pathogen causing life-threatening infections. Lung injury and the development of sepsis depend largely on the expression of type III secretion system (TTSS) virulence. TTSS functions as a molecular syringe to deliver toxins directly to the cytosol of cells, inhibit innate immune mechanisms, and prevent bacterial clearance. Polyclonal antibodies that bind to PcrV of P. aeruginosa inhibit the delivery of type III toxins and enhance the clearance of bacteria during acute lung infections. PcrV is a homologue of LcrV, a protective antigen in the Yersinia TTSS and an integral component of TTSS. In this study, a murine monoclonal antibody (MAb) to PcrV was generated: MAb 166, which is protective against P. aeruginosa when coinstilled with the bacterial inoculum or intraperitoneally transferred to mice. Fab fragments from MAb 166 prevent sepsis and death. The epitope bound by MAb 166 was mapped to the carboxyl-terminus of PcrV.

The prophylactic effects of human IgG derived from sera containing high anti-PcrV titers against pneumonia-causingPseudomonas aeruginosa

“…From our previous experience of screening for effective antibody clones or IgG extracted from hyper-immune serum that can block type III secretion-associated lung injury, we found that the pre-mixed setting produced the clearest direct effects with which to evaluate the efficacy of antibodies in blocking acute lung injury in infected animals. 14,20,23 In other words, if no protective effect was observed in the pre-mixed setting, administration of the antibodies via different routes (i.e., intravenous and intramuscular) would be unlikely to generate any protective effects. The effect of 10 human IgGs displaying high anti-PcrV titers (high titer hu-IgG) were compared with that of 10 human IgGs displaying low anti-PcrV titers (low titer hu-IgG), with saline used as a negative control and rabbitderived anti-PcrV polyclonal IgG (anti-PcrV rab-IgG) used as a positive control.…”

“…Our target is PcrV, which forms the tip of the type III secretion apparatus responsible for the delivery of type III secretory toxins (i.e., ExoS, ExoT, ExoU, and ExoY) into target cells. 12 Based on the preclinical data for the mAb166 murine anti-PcrV monoclonal antibody in various animal models, 19,20,22,39 a humaneered anti-PcrV monoclonal antibody (KB001-A, KaloBios Pharmaceuticals) has been developed 23 and tested for treating P. aeruginosa pneumonia in patients with cystic fibrosis and in mechanically ventilated patients in Phase II studies in the United States 24 and France. 25 Although Figure 10.…”

“…10,13,14 Active immunization with recombinant PcrV protects animals from lethal P. aeruginosa infections [14][15][16] , and anti-PcrV antibodies also protect infected animals from acute lung injury, bacteremia, and sepsis. 14,[17][18][19][20][21][22] Based on these experimental results, an engineered human anti-PcrV antibody was tested in patients in Phase II trials [23][24][25] , but no therapies based on it have been adapted for clinical use as yet.…”

“…For the effective blockade of type III secretion-associated virulence, for example, a monoclonal antibody needs to bind a specific blocking epitope region on the PcrV molecule. 20 This means that a high serum titer against PcrV may not always correlate with an effective blocking capability against type III secretion. Therefore, in this study, we extracted IgG fractions from human sera that possessed high or low anti-PcrV titers in our epidemiological survey.…”

FightingPseudomonas aeruginosainfection in patients with cystic fibrosis: orphan drug designation for a novel PcrV antibody

“…The TTSS is a complex composed of approximately 30 different proteins, making it one of the most complex secretion systems in nature. The role of TTSS has been validated as a target for Pa virulence in animal studies of acute Pa infection [8] using antibodies directed against Pa PcrV. PcrV is a structural translocation protein located at the tip of the TTSS, and secretion of PcrV enables intracellular delivery of a number of cytotoxic proteins, which interfere with host-cell signal transduction mechanisms, which result in either attenuation of the host's ability to orchestrate an immune response or an exaggerated pro-inflammatory response, and both processes may include cell death [9,10].…”