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In Vivo Toxicity and Immunological Characterization of Detoxified Recombinant Botulinum Neurotoxin Type A

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Abstract

Purpose

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.

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Abbreviations

ABSL-3:

Animal Biosafety Level 3

BAL:

Broncho-alveolar lavage

BCA:

Bicinchoninic acid protein assay

Bis-Tris:

[Bis (2-hydroxyethyl) imino-tris (hydroxymethyl) methane-HCl]

BoNT/A:

Full-length botulinum neurotoxin Type A

drBoNT/A:

Deactivated recombinant botulinum neurotoxin type A

eGFP:

Enhanced Green Fluorescent Protein

ELISA:

Enzyme-linked immunosorbent assay

FRET:

Fluorescence energy resonance transfer

HC:

Heavy Chain

i.p:

Intraperitoneal route

i.v:

Intravenous route

IgA:

Immunoglobulin A

IgG:

Immunoglobulin G

kDa:

kilodalton

LC:

Light Chain

LD50 :

A dose that proves lethal to 50% of the given population

PLGA (50:50):

Poly Lactic-co-Glycolic Acid biodegradable polymer

PVDF:

Polyvinyl difluoride membrane

rHc/A-BoNT/A:

Recombinant heavy chain of botulinum neurotoxin type A

rLC/A-BoNT/A:

Recombinant light chain of botulinum neurotoxin type A

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SNAP-25:

Synaptosomal-associated protein 25

Vitamin-E TPGS:

Vitamin – E ~ D -α-Tocopherol polyethylene glycol succinate

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ACKNOWLEDGMENTS AND DISCLOSURES

The study was supported in part by a NIAID (NIH) grants [1U01A1078070-01and R03AI103868] and a contract [HSHQDC-12-C-00071] from the Department of Homeland Security through Omni Array Biotechnology. I would like to sincerely thank Mr. Paul Lindo, Ms. Koyel Ghosal and Ms. Harkiran Dhaliwal for being part of animal husbandry team.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, Massachusetts, 02747, USA

    Easwaran Ravichandran, Pavithra Janardhanan, Kruti Patel, Stephen Riding, Shuowei Cai & Bal Ram Singh

  2. Botulinum Research Center, Institute of Advanced Sciences, Dartmouth, Massachusetts, 02747, USA

    Bal Ram Singh

  3. Prime Bio, Inc., Dartmouth, Massachusetts, 02747, USA

    Bal Ram Singh

  4. Aurobindo Pharma USA Inc, 6 Wheeling Road, Dayton, New Jersey, 08810, USA

    Easwaran Ravichandran

Authors
  1. Easwaran Ravichandran
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  2. Pavithra Janardhanan
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  3. Kruti Patel
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  4. Stephen Riding
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  5. Shuowei Cai
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  6. Bal Ram Singh
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Correspondence to Bal Ram Singh.

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Ravichandran, E., Janardhanan, P., Patel, K. et al. In Vivo Toxicity and Immunological Characterization of Detoxified Recombinant Botulinum Neurotoxin Type A. Pharm Res 33, 639–652 (2016). https://doi.org/10.1007/s11095-015-1816-x

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  • DOI: https://doi.org/10.1007/s11095-015-1816-x

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