title: In situ gastrointestinal protection against anthrax edema toxin by single-chain antibody fragment producing lactobacilli.
abstract: BACKGROUND
  Anthrax is caused by the bacterium Bacillus anthracis and is regarded as one of the most prominent bioterrorism threats. Anthrax toxicity is induced by the tripartite toxin complex, composed of the receptor-binding anthrax protective antigen and the two enzymatic subunits, lethal factor and edema factor. Recombinant lactobacilli have previously been used to deliver antibody fragments directed against surface epitopes of a variety of pathogens, including Streptococcus mutans, Porphyromonas gingivalis, and rotavirus. Here, we addressed whether or not anthrax toxins could be targeted and neutralised in the gastrointestinal tract by lactobacilli producing recombinant antibody fragments as a model system for toxin neutralisation in the gastrointestinal lumen.
  
  
  RESULTS
  The neutralising anti-PA scFv, 1H, was expressed in L. paracasei as a secreted protein, a cell wall-anchored protein or both secreted and wall-anchored protein. Cell wall display on lactobacilli and PA binding of the anchored constructs was confirmed by flow cytometry analysis. Binding of secreted or attached scFv produced by lactobacilli to PA were verified by ELISA. Both construct were able to protect macrophages in an in vitro cytotoxicity assay. Finally, lactobacilli producing the cell wall attached scFv were able to neutralise the activity of anthrax edema toxin in the GI tract of mice, in vivo.
  
  
  CONCLUSIONS
  We have developed lactobacilli expressing a neutralising scFv fragment against the PA antigen of the anthrax toxin, which can provide protection against anthrax toxins both in vitro and in vivo. Utilising engineered lactobacilli therapeutically for neutralising toxins in the gastrointestinal tract can potential be expanded to provide protection against a range of additional gastrointestinal pathogens. The ability of lactobacilli to colonise the gastrointestinal tract may allow the system to be used both prophylactically and therapeutically.
authors.affiliation: Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden.
authors.forename: Kasper Krogh
authors.initials: KK
authors.lastname: Andersen
authors.forename: Harold
authors.initials: H
authors.lastname: Marcotte
authors.forename: Beatriz
authors.initials: B
authors.lastname: Álvarez
authors.forename: Prosper N
authors.initials: PN
authors.lastname: Boyaka
authors.forename: Lennart
authors.initials: L
authors.lastname: Hammarström
mesh_terms: D000818:Animals; D000881:Anthrax / Q000276:immunology / Q000517:prevention & control*; D000907:Antibodies, Bacterial / Q000096:biosynthesis*; D057134:Antibodies, Neutralizing / Q000096:biosynthesis; D000942:Antigens, Bacterial / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D002460:Cell Line; D005260:Female; D041981:Gastrointestinal Tract / Q000276:immunology / Q000382:microbiology; D005818:Genetic Engineering; D007778:Lactobacillus / Q000235:genetics / Q000276:immunology*; D008264:Macrophages / Q000276:immunology / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids; D057127:Single-Chain Antibodies / Q000096:biosynthesis*
chemical_list: D000907:Antibodies, Bacterial; D057134:Antibodies, Neutralizing; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D057127:Single-Chain Antibodies; C030325:anthrax toxin