ABSTRACTPseudomonas aeruginosais 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 theP. aeruginosatype 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 targetingP. 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 T3SSin vitrofailed to provide protection in a mouse model ofP. aeruginosainfection, suggesting that effectivein vivoinhibition of T3SS with anti-PcrV MAbs is epitope dependent. V2L2MD, while not the most potent MAb as assessed byin vitrocytotoxicity inhibition assays, provided strong prophylactic protection in several murine infection models and a postinfection therapeutic model. V2L2MD mediated significantly (P< 0.0001) betterin vivoprotection 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 ofP. aeruginosainfections in high-risk populations.