Skip to main content
SpringerLink
Log in

In Vivo Neutralization of Botulinum Neurotoxins Serotype E with Heavy-chain Camelid Antibodies (VHH)

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Ingestion of botulinum neurotoxin (BoNT) results in botulism, a severe and frequent fatal disease known in the world. Current treatments rely on antitoxins, such as equine antitoxin and human botulism immunoglobulin. In some cases, side effects have been reported, including early anaphylactic shock and late serum sickness. Thus, diagnosis and treatment measure of BoNT are necessary and crucial. In the present study, a single-domain variable heavy-chain (VHH) antibody fragment was obtained from an immune dromedary phage display library against the putative binding domain of botulinum neurotoxin E (BoNT/E), a non-toxic 50-kDa fragment. The characteristics of nanobody VHH include excellent production, superior heat stability and specific binding capacity to soluble antigen without cross-reaction to other relevant or irrelevant antigens. A total of 150 ng/Kg of nanobody entirely neutralized 3LD50 of the BoNT/E in an in vivo challenge of the mice. This phenomenon indicates BoNT/E toxin neutralizing capacity of the produced nanobody. These results also suggest possession of unique properties by the nanobody applicable in diagnostics or therapeutic purposes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Arbabi Ghahroudi, M., Desmyter, A., Wyns, L., Hamers, R., & Muyldermans, S. (1997). Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Letters, 414, 521–526.

    Article  CAS  Google Scholar 

  2. Basiri, M., Mousavi, S. L., Basiri, H., & Rasooli, I. (2010). An epitopic approach to designing and characterization of a multiple antigenic polypeptide against Botulinum neurotoxins A and E. World Journal of Microbiology & Biotechnology, 26, 1659–1666.

    Article  CAS  Google Scholar 

  3. Beatty, J. D., Beatty, B. G., & Vlahos, W. G. (1987). Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. Journal of Immunological Methods, 100, 173–179.

    Article  CAS  Google Scholar 

  4. Behar, G., Siberil, S., Groulet, A., Chames, P., Pugniere, M., Boix, C., et al. (2008). Isolation and characterization of anti-FcγRIII (CD16) llama single-domain antibodies that activate natural killer cells. Protein Engineering, Design & Selection, 21, 1–10.

    Article  CAS  Google Scholar 

  5. Black, R. E., & Gunn, R. A. (1980). Hypersensitivity reactions associated with botulinal antitoxin. The American journal of medicine, 69, 567–570.

    Article  CAS  Google Scholar 

  6. Conrath, K. E., Lauwereys, M., Galleni, M., Matagne, A., Frere, J. M., Kinne, J., et al. (2001). β-Lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae. Antimicrobial Agents and Chemotherapy, 45, 2807.

    Article  CAS  Google Scholar 

  7. Devaux, C., Moreau, E., Goyffon, M., Rochat, H., & Billiald, P. (2001). Construction and functional evaluation of a single chain antibody fragment that neutralizes toxin AahI from the venom of the scorpion Androctonus australis hector. European Journal of Biochemistry, 268, 694–702.

    Article  CAS  Google Scholar 

  8. Dolly, J. O., Black, J., Williams, R. S., & Melling, J. (1984). Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization.

  9. Dong, J., Thompson, A. A., Fan, Y., Lou, J., Conrad, F., Ho, M., et al. (2010). A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic [alpha]-exosite binding region. Journal of Molecular Biology, 397, 1106–1118.

    Article  CAS  Google Scholar 

  10. Doyle, P. J., Arbabi-Ghahroudi, M., Gaudette, N., Furzer, G., Savard, M. E., Gleddie, S., et al. (2008). Cloning, expression, and characterization of a single-domain antibody fragment with affinity for 15-acetyl-deoxynivalenol. Molecular Immunology, 45, 3703–3713.

    Article  CAS  Google Scholar 

  11. Dressler, D., & Adib Saberi, F. (2005). Botulinum toxin: Mechanisms of action. European Neurology, 53, 3–9.

    Article  CAS  Google Scholar 

  12. Dumoulin, M., Conrath, K., Van Meirhaeghe, A., Meersman, F., Heremans, K., Frenken, L. G. J., et al. (2002). Single-domain antibody fragments with high conformational stability. Protein Science, 11, 500–515.

    Article  CAS  Google Scholar 

  13. Ebrahimizadeh, W., Mousavi Gargari, S., Rajabibazl, M., Safaee Ardekani, L., Zare, H. & Bakherad, H. (2012). Isolation and characterization of protective anti-LPS nanobody against V. cholerae O1 recognizing Inaba and Ogawa serotypes. Applied Microbiology and Biotechnology 1–10.

  14. Frenken, L. G. J., van der Linden, R. H. J., Hermans, P. W. J. J., Bos, J. W., Ruuls, R. C., de Geus, B., et al. (2000). Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae. Journal of Biotechnology, 78, 11–21.

    Article  CAS  Google Scholar 

  15. Gargari, S. L. M., Rasooli, I., Valipour, E., Basiri, M., Nazarian, S., Amani, J., & Farhadi, N. (2011). Immunogenic and protective potentials of recombinant receptor binding domain and a C-terminal fragment of Clostridium botulinum neurotoxin type E. Iranian Journal of Biotechnology (IJB) 9.

  16. Gessler, F., Hampe, K., Schmidt, M., & Bohnel, H. (2006). Immunomagnetic beads assay for the detection of botulinum neurotoxin types C and D. Diagnostic Microbiology and Infectious Disease, 56, 225–232.

    Article  CAS  Google Scholar 

  17. Gill, D. M. (1982). Bacterial toxins: A table of lethal amounts. Microbiology and Molecular Biology Reviews, 46, 86.

    CAS  Google Scholar 

  18. Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hammers, C., Songa, E. B., et al. (1993). Naturally occurring antibodies devoid of light chains. Nature, 363, 446–448.

    Article  CAS  Google Scholar 

  19. Harmsen, M. M., & De Haard, H. J. (2007). Properties, production, and applications of camelid single-domain antibody fragments. Applied Microbiology and Biotechnology, 77, 13–22.

    Article  CAS  Google Scholar 

  20. Harmsen, M. M., Van Solt, C. B., Van Zijderveld-van Bemmel, A., Niewold, T. A., & Van Zijderveld, F. G. (2006). Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy. Applied Microbiology and Biotechnology, 72, 544–551.

    Article  CAS  Google Scholar 

  21. Hayhurst, A., & Georgiou, G. (2001). High-throughput antibody isolation. Current Opinion in Chemical Biology, 5, 683–689.

    Article  CAS  Google Scholar 

  22. Hibbs, R. G., Weber, J. T., Corwin, A., Allos, B. M., El Rehim, M. S. A., El Sharkawy, S., et al. (1996). Experience with the use of an investigational F (ab’) 2 heptavalent botulism immune globulin of equine origin during an outbreak of type E botulism in Egypt. Clinical Infectious Diseases, 23, 337.

    Article  CAS  Google Scholar 

  23. Hmila, I., Abdallah, R., Saerens, D., Benlasfar, Z., Conrath, K., Ayeb, M. E., et al. (2008). VHH, bivalent domains and chimeric Heavy chain-only antibodies with high neutralizing efficacy for scorpion toxin AahI’. Molecular Immunology, 45, 3847–3856.

    Article  CAS  Google Scholar 

  24. Jones, R. G. A., Corbel, M. J., & Sesardic, D. (2006). A review of WHO International Standards for botulinum antitoxins. Biologicals, 34, 223–226.

    Article  CAS  Google Scholar 

  25. Kastelic, D., Frkovic-Grazio, S., Baty, D., Truan, G., Komel, R., & Pompon, D. (2009). A single-step procedure of recombinant library construction for the selection of efficiently produced llama VH binders directed against cancer markers. Journal of Immunological Methods, 350, 54–62.

    Article  CAS  Google Scholar 

  26. Lacy, D. B., Tepp, W., Cohen, A. C., DasGupta, B. R., & Stevens, R. C. (1998). Crystal structure of botulinum neurotoxin type A and implications for toxicity. Nature Structural & Molecular Biology, 5, 898–902.

    Article  CAS  Google Scholar 

  27. Ladenson, R. C., Crimmins, D. L., Landt, Y., & Ladenson, J. H. (2006). Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragment. Analytical Chemistry, 78, 4501–4508.

    Article  CAS  Google Scholar 

  28. Lalli, G., Herreros, J., Osborne, S. L., Montecucco, C., Rossetto, O., & Schiavo, G. (1999). Functional characterisation of tetanus and botulinum neurotoxins binding domains. Journal of Cell Science, 112, 2715–2724.

    CAS  Google Scholar 

  29. Ligler, F. S., Taitt, C. R., Shriver-Lake, L. C., Sapsford, K. E., Shubin, Y., & Golden, J. P. (2003). Array biosensor for detection of toxins. Analytical and Bioanalytical Chemistry, 377, 469–477.

    Article  CAS  Google Scholar 

  30. Mahrhold, S., Rummel, A., Bigalke, H., Davletov, B., & Binz, T. (2006). The synaptic vesicle protein 2C mediates the uptake of botulinum neurotoxin A into phrenic nerves. FEBS Letters, 580, 2011–2014.

    Article  CAS  Google Scholar 

  31. Maynard, J., & Georgiou, G. (2000). Antibody engineering. Annual Review of Biomedical Engineering, 2, 339–376.

    Article  CAS  Google Scholar 

  32. Mousli, M., Devaux, C., Rochat, H., Goyffon, M., & Billiald, P. (1999). A recombinant single-chain antibody fragment that neutralizes toxin II from the venom of the scorpion Androctonus australis hector. FEBS Letters, 442, 183–188.

    Article  CAS  Google Scholar 

  33. Muyldermans, S. (2001). Single domain camel antibodies: current status. Reviews in Molecular Biotechnology, 74, 277–302.

    Article  CAS  Google Scholar 

  34. Muyldermans, S., Atarhouch, T., Saldanha, J., Barbosa, J., & Hamers, R. (1994). Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Engineering, 7, 1129.

    Article  CAS  Google Scholar 

  35. Nguyen, V. K., Hamers, R., Wyns, L., & Muyldermans, S. (2000). Camel heavy-chain antibodies: diverse germline VHH and specific mechanisms enlarge the antigen-binding repertoire. The EMBO Journal, 19, 921–930.

    Article  CAS  Google Scholar 

  36. Rivera, V. R., Gamez, F. J., Keener, W. K., White, J. A., & Poli, M. A. (2006). Rapid detection of Clostridium botulinum toxins A, B, E, and F in clinical samples, selected food matrices, and buffer using paramagnetic bead-based electrochemiluminescence detection. Analytical Biochemistry, 353, 248–256.

    Article  CAS  Google Scholar 

  37. Sambrook, J., & Russell, D. W. (2001). Molecular cloning: a laboratory manual. CSHL Press.

  38. Sharma, S. K., & Whiting, R. C. (2005). Methods for detection of Clostridium botulinum toxin in foods. Journal of Food Protection&# 174, 68, 1256–1263.

    Google Scholar 

  39. Sharma, S. K., Ferreira, J. L., Eblen, B. S., & Whiting, R. C. (2006). Detection of type A, B, E, and F Clostridium botulinum neurotoxins in foods by using an amplified enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies. Applied and Environmental Microbiology, 72, 1231–1238.

    Article  CAS  Google Scholar 

  40. Simpson, L. L. (1980). Kinetic studies on the interaction between botulinum toxin type A and the cholinergic neuromuscular junction. Journal of Pharmacology and Experimental Therapeutics, 212, 16.

    CAS  Google Scholar 

  41. Simpson, L. L. (1981). The origin, structure, and pharmacological activity of botulinum toxin. Pharmacological Reviews, 33, 155–188.

    CAS  Google Scholar 

  42. Simpson, L. L. (1990). The study of clostridial and related toxins. The search for unique mechanisms and common denominators. Journal de physiologie, 84, 143.

    CAS  Google Scholar 

  43. Swain, M. D., Anderson, G. P., Zabetakis, D., Bernstein, R. D., Liu, J. L., Sherwood, L. J., et al. (2010). Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin. Analytical and Bioanalytical Chemistry, 398, 339–348.

    Article  CAS  Google Scholar 

  44. Tremblay, J. M., Kuo, C. L., Abeijon, C., Sepulveda, J., Oyler, G., Hu, X., et al. (2010). Camelid single domain antibodies (VHHs) as neuronal cell intrabody binding agents and inhibitors of Clostridium botulinum neurotoxin (BoNT) proteases. Toxicon, 56, 990–998.

    Article  CAS  Google Scholar 

  45. Vermeer, A. W. P., & Norde, W. (2000). The thermal stability of immunoglobulin: unfolding and aggregation of a multi-domain protein. Biophysical Journal, 78, 394–404.

    Article  CAS  Google Scholar 

  46. Vu, K. B., Ghahroudi, M. A., Wyns, L., & Muyldermans, S. (1997). Comparison of llama VH sequences from conventional and heavy chain antibodies. Molecular Immunology, 34, 1121–1131.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, Shahed University, Tehran-Qom Express Way, Opposite Imam Khomeini’s Shrine, 3319118651, Tehran, Iran

    Hamid Bakherad, Seyed Latif Mousavi Gargari, Iraj Rasooli, Walead Ebrahimizadeh, Leila Safaee Ardakani & Hamed Zare

  2. Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Masoumeh RajabiBazl

  3. Department of Biology, Basic Science Faculty, Shahid Chamran University, Ahvaz, Iran

    Mohammad Mohammadi

Authors
  1. Hamid Bakherad
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Seyed Latif Mousavi Gargari
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Iraj Rasooli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Masoumeh RajabiBazl
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mohammad Mohammadi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Walead Ebrahimizadeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Leila Safaee Ardakani
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hamed Zare
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Seyed Latif Mousavi Gargari.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 4205 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bakherad, H., Mousavi Gargari, S.L., Rasooli, I. et al. In Vivo Neutralization of Botulinum Neurotoxins Serotype E with Heavy-chain Camelid Antibodies (VHH). Mol Biotechnol 55, 159–167 (2013). https://doi.org/10.1007/s12033-013-9669-1

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-013-9669-1

Keywords

Search

Navigation