title: In Vivo Toxicity and Immunological Characterization of Detoxified Recombinant Botulinum Neurotoxin Type A.
abstract: OBJECTIVE
  A double-mutant E224A/E262A full-length botulinum neurotoxin (BoNT) Type A with structural similarity to native BoNT/A but lacking the endopeptidase activity provides an ideal surrogate for testing pharmacokinetics and immunochemical characteristics of BoNT.
  
  
  METHODS
  We determined lethality (LD50) of deactivated recombinant botulinum neurotoxin (drBoNT/A) to be 24.0 μg by intraperitoneal route (i.p). The polypeptide drBoNT/A labeled with near infra-red dye 800 (NIR 800) was used to examine its distribution to different organs using whole body imaging when administered to mice via intravenous (i.v) or i.p route. Also, drBoNT/A was used to evaluate its immunogenicity in Balb/C mice model.
  
  
  RESULTS
  drBoNT/A was found to be highly immunogenic when tested under various in vivo conditions in Balb/C mice model. For the first time we have demonstrated that a full length 150 kDa drBoNT/A, by administering via inhalation route in mice model, has evoked both circulating immunoglobulin levels of IgG and secretory IgA at the mucosal surface. The immunoglobulin levels were sufficient enough to protect against the challenge dose of native BoNT toxin in mice model. Tissue distribution of drBoNT/A seems to be similar to that of native toxin.
  
  
  CONCLUSIONS
  Based on the characteristics described in this report this nontoxic holotoxin protein will assist us to explore the window of opportunity available for therapeutic treatment in case of unnatural poisoning, and also it can be an effective vaccine candidate.
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, Massachusetts, 02747, USA.|Aurobindo Pharma USA Inc, 6 Wheeling Road, Dayton, New Jersey, 08810, USA.
authors.forename: Easwaran
authors.initials: E
authors.lastname: Ravichandran
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, Massachusetts, 02747, USA.
authors.forename: Pavithra
authors.initials: P
authors.lastname: Janardhanan
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, Massachusetts, 02747, USA.
authors.forename: Kruti
authors.initials: K
authors.lastname: Patel
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, Massachusetts, 02747, USA.
authors.forename: Stephen
authors.initials: S
authors.lastname: Riding
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, Massachusetts, 02747, USA.
authors.forename: Shuowei
authors.initials: S
authors.lastname: Cai
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, Massachusetts, 02747, USA. bsingh@inads.org.|Botulinum Research Center, Institute of Advanced Sciences, Dartmouth, Massachusetts, 02747, USA. bsingh@inads.org.|Prime Bio, Inc., Dartmouth, Massachusetts, 02747, USA. bsingh@inads.org.
authors.forename: Bal Ram
authors.initials: BR
authors.lastname: Singh
mesh_terms: D000818:Animals; D000917:Antibody Formation / Q000276:immunology*; D019274:Botulinum Toxins, Type A / Q000276:immunology*; D045744:Cell Line, Tumor; D005260:Female; D007071:Immunoglobulin A, Secretory / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000276:immunology*; D014018:Tissue Distribution / Q000276:immunology
chemical_list: D007071:Immunoglobulin A, Secretory; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D019274:Botulinum Toxins, Type A
keywords: Clostridium; botulinum; deactivated neurotoxin; drug delivery; microsphere; mucosal vaccine; toxicity; vaccine