Abstract
Tetanus toxin (TeNT) and botulinum neurotoxins (BoNTs) are neuroprotein toxins, with the latter being the most toxic known protein. They are structurally similar and contain three functional domains: an N-terminal catalytic domain (light chain), an internal heavy-chain translocation domain (HN domain), and a C-terminal heavy chain receptor binding domain (Hc domain or RBD). In this study, fusion functional domain molecules consisting of the TeNT RBD (THc) and the BoNT/A RBD (AHc) (i.e., THc-Linker-AHc and AHc-Linker-THc) were designed, prepared, and identified. The interaction of each Hc domain and the ganglioside receptor (GT1b) or the receptor synaptic vesicle glycoprotein 2 (SV2) was explored in vitro. Their immune response characteristics and protective efficacy were investigated in animal models. The recombinant THc-linker-AHc and AHc-linker-THc proteins with the binding activity had the correct size and structure, thus representing novel subunit vaccines. THc-linker-AHc and AHc-linker-THc induced high levels of specific neutralizing antibodies, and showed strong immune protective efficacy against both toxins. The high antibody titers against the two novel fusion domain molecules and against individual THc and AHc suggested that the THc and AHc domains, as antigens in the fusion functional domain molecules, do not interact with each other and retain their full key epitopes responsible for inducing neutralizing antibodies. Thus, the recombinant THc-linker-AHc and AHc-linker-THc molecules are strong and effective bivalent biotoxin vaccines, protecting against two biotoxins simultaneously. Our experimental design will be valuable to develop recombinant double-RBD fusion molecules as potent bivalent subunit vaccines against bio-toxins.
Key points
• Double-RBD fusion molecules from two toxins had the correct structure and activity.
• THc-linker-AHc and AHc-linker-THc efficiently protected against both biotoxins.
• Such bivalent biotoxin vaccines based on the RBD are a valuable experimental design.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
Alsarraf H, Dedic E, Bjerrum MJ, Østergaard O, Kristensen MP, Petersen JW, Jørgensen R (2017) Biophysical comparison of diphtheria and tetanus toxins with the formaldehyde-detoxified toxoids, the main components of diphtheria and tetanus vaccines. Virulence 8(8):1880–1889. https://doi.org/10.1080/21505594.2017.1321726
Avril A (2017) Therapeutic antibodies for biodefense. Adv Exp Med Biol 1053:173–205. https://doi.org/10.1007/978-3-319-72077-7_9
Baldwin MR, Tepp WH, Przedpelski A, Pier CL, Bradshaw M, Johnson EA, Barbieri JT (2008) Subunit vaccine against the seven serotypes of botulism. Infect Immun 76(3):1314–1318. https://doi.org/10.1128/iai.01025-07
Bayart C, Peronin S, Jean E, Paladino J, Talaga P, Borgne ML (2017) The combined use of analytical tools for exploring tetanus toxin and tetanus toxoid structures. J Chromatogr B Analyt Technol Biomed Life Sci 1054:80–92. https://doi.org/10.1016/j.jchromb.2017.04.009
Chai P, Pu X, Ge J, Ren S, Xia X, Luo A, Wang S, Wang X, Li J (2021) The recombinant protein combined vaccine based on the fragment C of tetanus toxin and the cross-reacting material 197. Appl Microbiol Biotechnol 105(4):1683–1692. https://doi.org/10.1007/s00253-021-11139-8
Connan C, Popoff MR (2017) Uptake of clostridial neurotoxins into cells and dissemination. Curr Top Microbiol Immunol 406:39–78. https://doi.org/10.1007/82_2017_50
Dong M, Yeh F, Tepp WH, Dean C, Johnson EA, Janz R, Chapman ER (2006) SV2 is the protein receptor for botulinum neurotoxin A. Science 312(5773):592–596. https://doi.org/10.1126/science.1123654
Dong M, Masuyer G, Stenmark P (2019) Botulinum and tetanus neurotoxins. Annu Rev Biochem 88:811–837. https://doi.org/10.1146/annurev-biochem-013118-111654
Fotinou C, Emsley P, Black I, Ando H, Ishida H, Kiso M, Sinha KA, Fairweather NF, Isaacs NW (2001) The crystal structure of tetanus toxin Hc fragment complexed with a synthetic GT1b analogue suggests cross-linking between ganglioside receptors and the toxin. J Biol Chem 276(34):32274–32281. https://doi.org/10.1074/jbc.M103285200
Ghotloo S, Golsaz-Shirazi F, Amiri MM, Jeddi-Tehrani M, Shokri F (2020) Epitope mapping of tetanus toxin by monoclonal antibodies: implication for immunotherapy and vaccine design. Neurotox Res 37(2):239–249. https://doi.org/10.1007/s12640-019-00096-w
Harper CB, Martin S, Nguyen TH, Daniels SJ, Lavidis NA, Popoff MR, Hadzic G, Mariana A, Chau N, McCluskey A, Robinson PJ, Meunier FA (2011) Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism. J Biol Chem 286(41):35966–35976. https://doi.org/10.1074/jbc.M111.283879
Karalewitz AP, Barbieri JT (2012) Vaccines against botulism. Curr Opin Microbiol 15(3):317–324. https://doi.org/10.1016/j.mib.2012.05.009
Keller JE (2008) Characterization of new formalin-detoxified botulinum neurotoxin toxoids. Clin Vaccine Immunol 15(9):1374–1379. https://doi.org/10.1128/cvi.00117-08
Li Z, Lu JS, Liu S, Wang R, Xu Q, Yu YZ, Yang ZX (2021) Recombinant L-HN fusion antigen derived from the L and HN domains of botulinum neurotoxin B stimulates a protective antibody response against active neurotoxin. Neurotox Res 39(4):1044–1053. https://doi.org/10.1007/s12640-021-00337-x
Liu FJ, Shi DY, Li ZY, Lu JS, Wang R, Pang XB, Yang ZX, Yu YZ (2020a) Evaluation of a recombinant tetanus toxin subunit vaccine. Toxicon 187:75–81. https://doi.org/10.1016/j.toxicon.2020.08.001
Liu FJ, Shi DY, Mao YY, Xiong XH, Lu JS, Pang XB, Dong XJ, Yang ZX, Yu YZ (2020b) Immunological characterisation and immunoprotective efficacy of functional domain antigens of botulinum neurotoxin serotype A. Vaccine 38(14):2978–2983. https://doi.org/10.1016/j.vaccine.2020.02.060
Lonati D, Schicchi A, Crevani M, Buscaglia E, Scaravaggi G, Maida F, Cirronis M, Petrolini VM, Locatelli CA (2020) Foodborne botulism: clinical diagnosis and medical treatment. Toxins (Basel) 12(8):509. https://doi.org/10.3390/toxins12080509
Maslanka SE, Lúquez C, Dykes JK, Tepp WH, Pier CL, Pellett S, Raphael BH, Kalb SR, Barr JR, Rao A, Johnson EA (2016) A novel botulinum neurotoxin, previously reported as serotype H, has a hybrid-like structure with regions of similarity to the structures of serotypes A and F and is neutralized with serotype A antitoxin. J Infect Dis 213(3):379–385. https://doi.org/10.1093/infdis/jiv327
Metz B, Michiels T, Uittenbogaard J, Danial M, Tilstra W, Meiring HD, Hennink WE, Crommelin DJA, Kersten GFA, Jiskoot W (2020) Identification of formaldehyde-induced modifications in diphtheria toxin. J Pharm Sci 109(1):543–557. https://doi.org/10.1016/j.xphs.2019.10.047
Middlebrook JL (2005) Production of vaccines against leading biowarfare toxins can utilize DNA scientific technology. Adv Drug Deliv Rev 57(9):1415–1423. https://doi.org/10.1016/j.addr.2005.01.016
Montecucco C (1986) How do tetanus and botulinum toxins bind to neuronal membranes? Trends Biochem Sci 11:314–317
Moreira GM, Cunha CE, Salvarani FM, Gonçalves LA, Pires PS, Conceição FR, Lobato FC (2014) Production of recombinant botulism antigens: a review of expression systems. Anaerobe 28:130–136. https://doi.org/10.1016/j.anaerobe.2014.06.003
O’Horo JC, Harper EP, El Rafei A, Ali R, DeSimone DC, Sakusic A, Abu Saleh OM, Marcelin JR, Tan EM, Rao AK, Sobel J, Tosh PK (2017) Efficacy of antitoxin therapy in treating patients with foodborne botulism: a systematic review and meta-analysis of cases, 1923–2016. Clin Infect Dis 66(suppl_1):S43-s56. https://doi.org/10.1093/cid/cix815
Pirazzini M, Rossetto O, Eleopra R, Montecucco C (2017) Botulinum neurotoxins: biology, pharmacology, and toxicology. Pharmacol Rev 69(2):200–235. https://doi.org/10.1124/pr.116.012658
Poulain B, Popoff MR (2019) Why are botulinum neurotoxin-producing bacteria so diverse and botulinum neurotoxins so toxic? Toxins (Basel) 11(1) https://doi.org/10.3390/toxins11010034
Quddus A, Luby S, Rahbar M, Pervaiz Y (2002) Neonatal tetanus: mortality rate and risk factors in Loralai District. Pakistan Int J Epidemiol 31(3):648–653. https://doi.org/10.1093/ije/31.3.648
Rai R, Singh DK (2012) Neonatal tetanus: a continuing challenge. Indian J Pediatr 79(12):1648–1650. https://doi.org/10.1007/s12098-011-0666-8
Ravichandran E, Al-Saleem FH, Ancharski DM, Elias MD, Singh AK, Shamim M, Gong Y, Simpson LL (2007) Trivalent vaccine against botulinum toxin serotypes A, B, and E that can be administered by the mucosal route. Infect Immun 75(6):3043–3054. https://doi.org/10.1128/iai.01893-06
Ravichandran E, Janardhanan P, Patel K, Riding S, Cai S, Singh BR (2016) In vivo toxicity and immunological characterization of detoxified recombinant botulinum neurotoxin type A. Pharm Res 33(3):639–652. https://doi.org/10.1007/s11095-015-1816-x
Rossetto O, Montecucco C (2019) Tables of toxicity of botulinum and tetanus neurotoxins. Toxins 11(12):686. https://doi.org/10.3390/toxins11120686
Rummel A (2013) Double receptor anchorage of botulinum neurotoxins accounts for their exquisite neurospecificity. Curr Top Microbiol Immunol 364:61–90. https://doi.org/10.1007/978-3-642-33570-9_4
Rummel A (2017) Two feet on the membrane: uptake of clostridial neurotoxins. Curr Top Microbiol Immunol 406:1–37. https://doi.org/10.1007/82_2016_48
Sharma S, Zhou Y, Singh BR (2006) Cloning, expression, and purification of C-terminal quarter of the heavy chain of botulinum neurotoxin type A. Protein Expr Purif 45(2):288–295. https://doi.org/10.1016/j.pep.2005.07.020
Shearer JD, Vassar ML, Swiderski W, Metcalfe K, Niemuth N, Henderson I (2010) Botulinum neurotoxin neutralizing activity of immune globulin (IG) purified from clinical volunteers vaccinated with recombinant botulinum vaccine (rBV A/B). Vaccine 28(45):7313–7318. https://doi.org/10.1016/j.vaccine.2010.08.076
Shearer JD, Manetz TS, House RV (2012) Preclinical safety assessment of recombinant botulinum vaccine A/B (rBV A/B). Vaccine 30(11):1917–1926. https://doi.org/10.1016/j.vaccine.2012.01.035
Shi DY, Chen BY, Mao YY, Zhou G, Lu JS, Yu YZ, Zhou XW, Sun ZW (2019) Development and evaluation of candidate subunit vaccine against botulinum neurotoxin serotype B. Hum Vaccin Immunother 15(3):755–760. https://doi.org/10.1080/21645515.2018.1547613
Shi DY, Liu FJ, Mao YY, Cui RT, Lu JS, Yu YZ, Dong XJ, Yang ZX, Sun ZW, Pang XB (2020) Development and evaluation of candidate subunit vaccine and novel antitoxin against botulinum neurotoxin serotype E. Hum Vaccin Immunother 16(1):100–108. https://doi.org/10.1080/21645515.2019.1633878
Shi DY, Liu FJ, Li ZY, Mao YY, Lu JS, Wang R, Pang XB, Yu YZ, Yang ZX (2022) Development and evaluation of a tetravalent botulinum vaccine. Hum Vaccin Immunother 18(5):2048621. https://doi.org/10.1080/21645515.2022.2048621
Shi DY, Lu JS, Mao YY, Liu FJ, Wang R, Du P, Yu S, Yu YZ, Yang ZX (2023) Characterization of a novel tetravalent botulism antitoxin based on receptor-binding domain of BoNTs. Appl Microbiol Biotechnol 107(10):3205–3216. https://doi.org/10.1007/s00253-023-12515-2
Sundeen G, Barbieri JT (2017) Vaccines against botulism. Toxins (Basel) 9(9):268. https://doi.org/10.3390/toxins9090268
Tehran D, Pirazzini M (2018) Novel botulinum neurotoxins: exploring underneath the iceberg tip. Toxins 10(5):190. https://doi.org/10.3390/toxins10050190
Thwaites CL, Beeching NJ, Newton CR (2015) Maternal and neonatal tetanus. Lancet 385(9965):362–370. https://doi.org/10.1016/s0140-6736(14)60236-1
Vessely C, Estey T, Randolph TW, Henderson I, Nayar R, Carpenter JF (2007) Effects of solution conditions and surface chemistry on the adsorption of three recombinant botulinum neurotoxin antigens to aluminum salt adjuvants. J Pharm Sci 96(9):2375–2389. https://doi.org/10.1002/jps.20880
Vessely C, Estey T, Randolph TW, Henderson I, Cooper J, Nayar R, Braun LJ, Carpenter JF (2009) Stability of a trivalent recombinant protein vaccine formulation against botulinum neurotoxin during storage in aqueous solution. J Pharm Sci 98(9):2970–2993. https://doi.org/10.1002/jps.21498
Webb RP, Smith LA (2013) What next for botulism vaccine development? Expert Rev Vaccines 12(5):481–492. https://doi.org/10.1586/erv.13.37
Yao G, Lam K-h, Perry K, Weisemann J, Rummel A, Jin R (2017) Crystal structure of the receptor-binding domain of botulinum neurotoxin type HA, also known as type FA or H. Toxins 9(3):93. https://doi.org/10.3390/toxins9030093
Yeh FL, Dong M, Yao J, Tepp WH, Lin G, Johnson EA, Chapman ER (2010) SV2 mediates entry of tetanus neurotoxin into central neurons. PLoS Pathog 6(11):e1001207. https://doi.org/10.1371/journal.ppat.1001207
Yu YZ, Li N, Wang RL, Zhu HQ, Wang S, Yu WY, Sun ZW (2008) Evaluation of a recombinant Hc of Clostridium botulinum neurotoxin serotype F as an effective subunit vaccine. Clin Vaccine Immunol 15(12):1819–1823. https://doi.org/10.1128/cvi.00239-08
Yu YZ, Li N, Zhu HQ, Wang RL, Du Y, Wang S, Yu WY, Sun ZW (2009) The recombinant Hc subunit of Clostridium botulinum neurotoxin serotype A is an effective botulism vaccine candidate. Vaccine 27(21):2816–2822. https://doi.org/10.1016/j.vaccine.2009.02.091
Yu YZ, Ma Y, Chen YX, Gong ZW, Wang S, Yu WY, Sun ZW (2011) Binding activity and immunogenic characterization of recombinant C-terminal quarter and half of the heavy chain of botulinum neurotoxin serotype A. Hum Vaccin 7(10):1090–1095. https://doi.org/10.4161/hv.7.10.16763
Funding
This work was partly supported by the Laboratory of Advanced Biotechnology Project (grant number BDZZ202204).
Author information
Authors and Affiliations
Contributions
Conceived and designed the experiments: XBP, YZY, and ZXY. Performed the experiments: BLL, JRW, XYL, and JSL. Analyzed the data: BLL, YZY, JSL, and FJL. Contributed reagents/materials/analysis tools: JRW, RW, PD, and SY. Wrote the paper: BLL, YZY, and JSL. All authors read and approved the final version of the manuscript.
Corresponding authors
Ethics declarations
Ethics approval
All animal experiments were approved by the Institution of Animal Care and Use Committee of the Beijing Institute of Biotechnology and performed with the permission of the Institute of Animal Care and Use Committee (IACUC) at the Academy of Military Medical Science, with the ethical approval number IACUC-DWZX-2021–014.
Disclaimer
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Li, BL., Wang, JR., Liu, XY. et al. Tetanus toxin and botulinum neurotoxin–derived fusion molecules are effective bivalent vaccines. Appl Microbiol Biotechnol 107, 7197–7211 (2023). https://doi.org/10.1007/s00253-023-12796-7
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-023-12796-7