Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B.
Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.
Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, Room 3C-38, 1001 Potrero Avenue, San Francisco, CA 94110, USA. frank.fan@ucsf.edu.
Yongfeng
Y
Fan
Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, Room 3C-38, 1001 Potrero Avenue, San Francisco, CA 94110, USA. jianbo.dong@ucsf.edu.
Jianbo
J
Dong
Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, Room 3C-38, 1001 Potrero Avenue, San Francisco, CA 94110, USA. jianlong.lou@ucsf.edu.
Jianlong
J
Lou
Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, Room 3C-38, 1001 Potrero Avenue, San Francisco, CA 94110, USA. wei.wen@ucsf.edu.
Weihua
W
Wen
Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, Room 3C-38, 1001 Potrero Avenue, San Francisco, CA 94110, USA. Conrad.fraser@ucsf.edu.
Fraser
F
Conrad
Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, Room 3C-38, 1001 Potrero Avenue, San Francisco, CA 94110, USA. isin.geren@medeniyet.edu.tr.
Isin N
IN
Geren
Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, Room 3C-38, 1001 Potrero Avenue, San Francisco, CA 94110, USA. MariaConsuelo.Garcia@ucsf.edu.
Consuelo
C
Garcia-Rodriguez
Molecular and Translational Sciences Division, United States Army Medical Institute of Infectious Diseases, Fort Detrick, MD 21702, USA. theresa.j.smith.civ@mail.mil.
Theresa J
TJ
Smith
Medical Countermeasures Technology, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702-5011, USA. Leonard.A.Smith1.civ@mail.mil.
Leonard A
LA
Smith
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. mho1@illinois.edu.
Mengfei
M
Ho
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. melalves@illinois.edu.
Melissa
M
Pires-Alves
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA. bawilson@life.illinois.edu.
Brenda A
BA
Wilson
Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco General Hospital, Room 3C-38, 1001 Potrero Avenue, San Francisco, CA 94110, USA. jim.marks@ucsf.edu.
James D
JD
Marks
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000992:Antitoxins / Q000276:immunology; D019274:Botulinum Toxins, Type A / Q000276:immunology / Q000633:toxicity*; D000939:Epitopes / Q000276:immunology; D005260:Female; D005434:Flow Cytometry; D020128:Inhibitory Concentration 50; D051379:Mice; D011487:Protein Conformation; D059748:Proteolysis; D050600:SNARE Proteins / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000992:Antitoxins; D000939:Epitopes; D050600:SNARE Proteins; D057127:Single-Chain Antibodies; C096323:rimabotulinumtoxinB; D019274:Botulinum Toxins, Type A
Botulinum neurotoxin serotype B; alpha-exosite; botulinum antitoxin; inhibitory antibodies