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Background: Botulinum neurotoxins (BoNTs) comprise seven agreed-on serotypes, A through G. In 2014, a novel chimeric neurotoxin produced by clostridial strain IBCA10-7060 was reported as BoNT/H, with subsequent names of BoNT/FA or BoNT/HA based on sequence homology of the N-terminus to BoNT/F, the C-terminus to BoNT/A and neutralization studies. The purpose of this study was to define the immunologic identity of the novel BoNT. Methods: monoclonal antibodies (mAbs) to the novel BoNT/H N-terminus were generated by antibody repertoire cloning and yeast display after immunization with BoNT/H LC-HN or BoNT/F LC-HN. Results: 21 unique BoNT/H LC-HN mAbs were obtained; 15 from the BoNT/H LC-HN immunized library (KD 0.78 nM to 182 nM) and six from the BoNT/F-immunized libraries (KD 20.5 nM to 1490 nM). A total of 15 of 21 mAbs also bound catalytically inactive BoNT/H holotoxin. The mAbs bound nine non-overlapping epitopes on the BoNT/H LC-HN. None of the mAbs showed binding to BoNT serotypes A-G, nor any of the seven subtypes of BoNT/F, except for one mAb that weakly bound BoNT/F5. Conclusions: The results, combined with the chimeric structure and neutralization by anti-A, but not anti-F antitoxin indicate that immunologically the novel BoNT is BoNT/HA. This determination has significant implications for existing countermeasures and potential vulnerabilities.
Yongfeng Fan; Jason R. Barash; Fraser Conrad; Jianlong Lou; Christina Tam; Luisa W. Cheng; Stephen S. Arnon; James D. Marks. The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA. Toxins 2019, 12, 9 .
AMA StyleYongfeng Fan, Jason R. Barash, Fraser Conrad, Jianlong Lou, Christina Tam, Luisa W. Cheng, Stephen S. Arnon, James D. Marks. The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA. Toxins. 2019; 12 (1):9.
Chicago/Turabian StyleYongfeng Fan; Jason R. Barash; Fraser Conrad; Jianlong Lou; Christina Tam; Luisa W. Cheng; Stephen S. Arnon; James D. Marks. 2019. "The Novel Clostridial Neurotoxin Produced by Strain IBCA10-7060 Is Immunologically Equivalent to BoNT/HA." Toxins 12, no. 1: 9.
Botulism, caused by exposure to one or more of the eight serotypes of botulinum neurotoxins (BoNTs) (BoNT/A through H), is often fatal without rapid treatment.
Jianlong Lou; James D. Marks. Botulinum Neurotoxins (BoNTs)—Antibody and Vaccine. Toxins 2018, 10, 495 .
AMA StyleJianlong Lou, James D. Marks. Botulinum Neurotoxins (BoNTs)—Antibody and Vaccine. Toxins. 2018; 10 (12):495.
Chicago/Turabian StyleJianlong Lou; James D. Marks. 2018. "Botulinum Neurotoxins (BoNTs)—Antibody and Vaccine." Toxins 10, no. 12: 495.
Human botulism is most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (KD). By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had KD values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633) is in pre-clinical development with an investigational new drug (IND) application filing expected in 2018.
Consuelo Garcia-Rodriguez; Ali Razai; Isin Geren; Jianlong Lou; Fraser Conrad; Wei-Hua Wen; Shauna Farr-Jones; Theresa J. Smith; Jennifer L. Brown; Janet C. Skerry; Leonard A. Smith; James D. Marks. A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes. Toxins 2018, 10, 105 .
AMA StyleConsuelo Garcia-Rodriguez, Ali Razai, Isin Geren, Jianlong Lou, Fraser Conrad, Wei-Hua Wen, Shauna Farr-Jones, Theresa J. Smith, Jennifer L. Brown, Janet C. Skerry, Leonard A. Smith, James D. Marks. A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes. Toxins. 2018; 10 (3):105.
Chicago/Turabian StyleConsuelo Garcia-Rodriguez; Ali Razai; Isin Geren; Jianlong Lou; Fraser Conrad; Wei-Hua Wen; Shauna Farr-Jones; Theresa J. Smith; Jennifer L. Brown; Janet C. Skerry; Leonard A. Smith; James D. Marks. 2018. "A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes." Toxins 10, no. 3: 105.
The standard of treatment for botulism, equine antitoxin, is a foreign protein with associated safety issues and a short serum half-life which excludes its use as a prophylactic antitoxin and makes it a less-than-optimal therapeutic. Due to these limitations, a recombinant monoclonal antibody (mAb) product is preferable. It has been shown that combining three mAbs that bind non-overlapping epitopes leads to highly potent botulinum neurotoxin (BoNT) neutralization. Recently, a triple human antibody combination for BoNT/A has demonstrated potent toxin neutralization in mouse models with no serious adverse events when tested in a Phase I clinical trial. However, a triple antibody therapeutic poses unique development and manufacturing challenges. Thus, potentially to streamline development of BoNT antitoxins, we sought to achieve the potency of multiple mAb combinations in a single IgG-based molecule that has a long serum half-life. The design, production, and testing of a single tri-epitopic IgG1-based mAb (TeAb) containing the binding sites of each of the three parental BoNT/A mAbs yielded an antibody of nearly equal potency to the combination. The approach taken here could be applied to the design and creation of other multivalent antibodies that could be used for a variety of applications, including toxin elimination.
Jianlong Lou; Weihua Wen; Fraser Conrad; Qi Meng; Jianbo Dong; Zhengda Sun; Consuelo Garcia-Rodriguez; Shauna Farr-Jones; Luisa W. Cheng; Thomas D. Henderson; Jennifer L. Brown; Theresa J. Smith; Leonard A. Smith; Anthony Cormier; James D. Marks. A Single Tri-Epitopic Antibody Virtually Recapitulates the Potency of a Combination of Three Monoclonal Antibodies in Neutralization of Botulinum Neurotoxin Serotype A. Toxins 2018, 10, 84 .
AMA StyleJianlong Lou, Weihua Wen, Fraser Conrad, Qi Meng, Jianbo Dong, Zhengda Sun, Consuelo Garcia-Rodriguez, Shauna Farr-Jones, Luisa W. Cheng, Thomas D. Henderson, Jennifer L. Brown, Theresa J. Smith, Leonard A. Smith, Anthony Cormier, James D. Marks. A Single Tri-Epitopic Antibody Virtually Recapitulates the Potency of a Combination of Three Monoclonal Antibodies in Neutralization of Botulinum Neurotoxin Serotype A. Toxins. 2018; 10 (2):84.
Chicago/Turabian StyleJianlong Lou; Weihua Wen; Fraser Conrad; Qi Meng; Jianbo Dong; Zhengda Sun; Consuelo Garcia-Rodriguez; Shauna Farr-Jones; Luisa W. Cheng; Thomas D. Henderson; Jennifer L. Brown; Theresa J. Smith; Leonard A. Smith; Anthony Cormier; James D. Marks. 2018. "A Single Tri-Epitopic Antibody Virtually Recapitulates the Potency of a Combination of Three Monoclonal Antibodies in Neutralization of Botulinum Neurotoxin Serotype A." Toxins 10, no. 2: 84.
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.
Yongfeng Fan; Jianbo Dong; Jianlong Lou; Weihua Wen; Fraser Conrad; Isin N. Geren; Consuelo Garcia-Rodriguez; Theresa J. Smith; Leonard A. Smith; Mengfei Ho; Melissa Pires-Alves; Brenda A. Wilson; James D. Marks. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B. Toxins 2015, 7, 3405 -3423.
AMA StyleYongfeng Fan, Jianbo Dong, Jianlong Lou, Weihua Wen, Fraser Conrad, Isin N. Geren, Consuelo Garcia-Rodriguez, Theresa J. Smith, Leonard A. Smith, Mengfei Ho, Melissa Pires-Alves, Brenda A. Wilson, James D. Marks. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B. Toxins. 2015; 7 (9):3405-3423.
Chicago/Turabian StyleYongfeng Fan; Jianbo Dong; Jianlong Lou; Weihua Wen; Fraser Conrad; Isin N. Geren; Consuelo Garcia-Rodriguez; Theresa J. Smith; Leonard A. Smith; Mengfei Ho; Melissa Pires-Alves; Brenda A. Wilson; James D. Marks. 2015. "Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B." Toxins 7, no. 9: 3405-3423.