We propose a novel antibacterial strategy directed at the neutralization of a critical bacterial virulence factor, csgA the monomeric subunit of curliated fimbriae. In addition to their classic role in bacterial adhesion and attachment, curli act as a virulence factor by virtue of their role in inducing the synthesis of inducible nitric oxide (NO) synthase (iNOS) derived NO. csgA deficient E. coli are less virulent and fail to induce severe hypotension in experimental models of septic shock. Our long-term objective is to market a human anti-csgA monoclonal antibody (MAb) that may be administered, in conjunction with standard antibiotic therapy, for the indication of severe E. coli infection. We intend to establish proof-of-principle that passive immunization with an anti-csgA MAb reduces inflammation and mortality in a rodent model of lethal E. coli pneumonia.
Aim #1 : Generate a series of murine anti-csgA monoclonal IgG Ab's. Mice will be immunized with recombinantly expressed His-tagged csgA. The resultant MAb-producing hybridomas will be screened for their affinity to recombinant csgA and to live E. coli. A total of 20 IgG-class anti-csgA murine MAb's will be selected and cloned.
Aim #2 : Rank order the in vivo potency of a series of murine anti-csgA MAb's. The selection of a lead anti-csgA MAb will be based upon a multi-step screening process. Task #1: we will utilize an LD100 murine model of pneumonia produced by direct intratracheal instillation of E. coli. MAb's will be administered after exposure to a lethal inoculum of E. coli and survival determined. 5 MAb's will be selected for further study on the basis of protection from mortality. Task #2: we will obtain a pharmacodynamic survival profile of the 5 lead anti-csgA MAb candidates. Task #3: we will establish a pharmacodynamic anti-inflammatory profile in a sublethal model of E. colipneumonia. Outcome measures will include body weight, neutrophil infiltration (myeloperoxidase), infectious burden (cfu), and lipid peroxidation (malondialdehyde). We expect to identify a murine anti-csgA MAb that provides potent protection against E. coli pneumonia induced lethality and pulmonary injury.
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