Clostridium difficile is the cause of pseudomembranous colitis and the leading cause of nosocomial intestinal infections in the U.S. It is responsible for substantial morbidity and has a significant economic impact on the health care system. C. difficile-associated disease (CDAD) is caused by antibiotics and there are currently no effective treatments for relapsing CDAD other than antibiotics. The overall goal of this project is to develop a novel, non-antibiotic treatment for CDAD that combines two promising therapeutic approaches. We will utilize non-pathogenic intestinal bacteria, which have shown clinical benefit in the treatment of CDAD, to deliver passive immunity against C. difficile toxins locally in the bowel. Specifically, we will create intestinal Lactobacillus strains capable of secreting neutralizing single chain antibodies (scFv) against C. difficile toxins A and B. Since a scFv against toxin B has already been developed by Meridian Bioscience, Inc., we will first engineer Lactobacillus to secrete this scFv and determine whether it is biologically active as a proof of concept for this approach. The specific aims of this Phase I study are therefore: 1. Express and characterize anti-toxin B scFv in intestinal Lactobacillus 2. Demonstrate that Lactobacillus-expressed scFv binds toxin B 3. Demonstrate that Lactobacillus-expressed scFv neutralizes toxin B cytotoxic activity Upon successful completion of this Phase I study, we will collaborate with Dr. John Morrow (Meridian Bioscience, Inc.) to produce a scFv against toxin A and express it in Lactobacillus. The efficacy of orally administered Lactobacillus strains secreting scFv against toxins A and B will then be tested in a well-established animal model of CDAD, the clindamycin-treated hamster. If the strains provide protection against CDAD in vivo, we will further optimize the affinity and expression levels of anti-toxin scFv, and develop Lactobacillus expression hosts suitable for administration to humans. These strains will contain the expression cassettes stably integrated into the bacterial genome, lack antibiotic resistance, and will not be able to survive outside the body. The genetically modified lactobacilli will then be manufactured and formulated in an oral dosage form for human clinical studies using Osel's proprietary technology.
Thesaurus Terms: Clostridium difficile, colitis, enteric bacteria, immunotherapy, oral administration, therapy design /development Lactobacillus, antitoxin, bacterial toxin, cytotoxicity, neutralizing antibody, passive immunization SDS polyacrylamide gel electrophoresis, enzyme linked immunosorbent assay, polymerase chain reaction
