Immunogenicity evaluation of rBoNT/E nanovaccine after mucosal administration

Document Type : Original Article


Biology Research Centre, Basic Science Faculty, Imam Hossein University, Tehran, Iran


Objective(s): The Botulism syndrome is caused by types A to G of botulinum neurotoxins. The binding domains of these neurotoxins are immunogenic and considered as appropriate candidate vaccines. Due to the low immunogenicity of recombinant vaccines, there have been many studies on the use of biocompatible carriers such as chitosan nanoparticles for the delivery of these vaccines. The aim of this study was evaluating the efficiency of chitosan nanoparticles as carriers for a candidate vaccine, binding domain of BoNT/E, through oral and intranasal routes.
Materials and Methods: Chitosan nanoparticles containing rBoNT/E binding domain, were synthesized via ionic gelation. After administration of the nanoparticles to mice through oral and intranasal routes, antibody titers were assessed by ELISA and, finally, all groups were challenged by active botulinum neurotoxin type E.
Results: The groups that received nanoparticles containing the antigen, through oral and intranasal routes, and the group that received the bare antigen orally, were able to tolerate 5×102 folds of MLD. The intranasally immunized mice by the bare antigen were able to tolerate 2×103 folds of the toxin’s MLD.
Conclusion: It seems that the use of chitosan nanoparticles has no significant effect on the protective immunization of the mice against botulinum BoNT/E in either route (P>0.05), even intranasal administration of the bare antigen gives better mice immunization against the toxin.


Main Subjects

1. Kobayashi R, Kohda T, Kataoka K, Ihara H, Kozaki S, Pascual DW, et al. A novel neurotoxoid vaccine prevents mucosal botulism. J Immunol 2005; 174:2190-2195.
2. Shone C,  Agostini H, Clancy J, Gu M, Yang HH, Chu Y, et al. Bivalent recombinant vaccine for botulinum neurotoxin types A and B based on a polypeptide comprising their effector and translocation domains that is protective against the predominant A and B subtypes. Infect Immun 2009; 77:2795-2801.
3. Byrne MP, Smith LA. Development of vaccines for prevention of botulism. Biochimie 2000; 82:955-966.
4. Henkel JS, Tepp WH, Przedpelski A, Fritz RB, Johnson EA, Barbieri JT. Subunit vaccine efficacy against Botulinum neurotoxin subtypes. Vaccine 2011; 29:7688-7695.
5. Ravichandran E, Al-Saleem FH, Ancharski DM, Elias MD, Singh AK, Shamim M, et al. Trivalent vaccine against botulinum toxin serotypes A, B, and E that can be administered by the mucosal route. Infect Immun 2007; 75:3043-3054.
6. Slütter B. Challenges and opportunities in nasal subunit vaccine delivery: mechanistic studies using ovalbumin as a model antigen. Thesis to obtain a PhD at Leiden University, 2011.
7. Tiyaboonchai W. Chitosan nanoparticles: a promising system for drug delivery. Naresuan Univ J 2003; 11:51-66.
8. Baudner BC, Balland O, Giuliani MM, Hoegen PV, Rappuoli R, Betbeder D, et al. Enhancement of protective efficacy following intranasal immunization with vaccine plus a nontoxic LTK63 mutant delivered with nanoparticles. Infect Immun 2002; 70:4785-4790.
9. Shahiwala A, Vyas TK, Amiji MM. Nanocarriers for systemic and mucosal vaccine delivery. recent patents on drug delivery & formulation 2007; 1:1-9.
10. Slütter B, Bal SM, Que I, Kaijzel E, Löwik C, Bouwstra J, et al. Antigen-adjuvant nano conjugates for nasal vaccination: an improvement over the use of nanoparticles? Mol Pharmaceutics 2010; 7:2207-2215.
11. Raut S, Sutar M, Singh S. Nasal vaccine: a novel approach in nasal drug delivery system. World J Pharm Pharm Sci 2015;4:1520-1536.
12. Hajizade A, Ebrahimi F, Salmanian AH, Arpanaei A, Amani J. Nanoparticles in vaccine development. J Appl Biotechnol Rep 2014; 1:125-134.
13. Ariful Islam M, Firdous J, Choi YJ, Yun CH, Cho CS. Design and application of chitosan microspheres as oral and nasal vaccine carriers: an updated review. Int J Nanomed 2012; 7:6077-6093.
14. Wang JJ, Zeng ZW, Xiao RZ, Xie T, Zhou GL, Zhan XR, et al. Recent advances of chitosan nanoparticles as drug carriers. Int J Nanomed 2011; 6:765-774.
15. Nagpal K, Singh SK, Mishra DN. Chitosan nanoparticles: a promising system in novel drug delivery. Chem. Pharm. Bull 2010; 58:1423-1430.
16. Zhao LM, Shi LE, Zhang ZL, Chen JM, Shi DD, Yang J, et al. Preparation and application of chitosan nanoparticles and nanofibers. Braz J Chem Eng 2011; 28:353-362.
17. Ferguson A, Humphreys KA, Croft NM. Technical report: results of immunological tests on fecal extracts are likely to be extremely misleading. Clin Exp Immunol 1995;99:70-75.
18. Valipour E, Moosavi ML, Amani J, Nazarian S. High level expression, purification and immunogenicity analysis of a protective recombinant protein against botulinum neurotoxin type E. World J Microbiol Biotechnol 2014; 30:1861-1867.
19. Zhao H, Nakamura K, Kohda T, Mukamoto M, Kozaki S. Characterization of the monoclonal antibody response to botulinum neurotoxin type A in the complexed and uncomplexed forms. Jpn J Infect Dis 2012; 65:138-145.
20. Baldwin MR, Tepp WH, Przedpelski A, Pier CL, Bradshaw M, Johnson EA, et al. Subunit vaccine against the seven serotypes of botulism. Infect Immun 2008; 76:1314-1318.
21. Mansour AA, Mousavi SL, Rasooli I, Nazarian S, Amani J, Farhadi N. Cloning, high level expression and immunogenicity of 1163-1256 residues of C-terminal heavy chain of C. botulinum neurotoxin type E. Biologicals 2010; 38:260-264.
22. Yu YZ, Sun ZW, Wang S, Yu WY. High-level expression of the Hc domain of Clostridium botulinum neurotoxin serotype A in Escherichia coli and its immunogenicity as an antigen. Sheng Wu Gong Cheng XueBao 2007; 23:812-817.
23. Boles J, West M, Montgomery V, Tammariello R, Pitt ML, Gibbs P, et al. Recombinant C fragment of botulinum neurotoxin B serotype (rBoNT/B (HC)) immune response and protection in the rhesus monkey. Toxicon 2006; 47:877-884.
24. Nazarian S, Gargari SL, Rasooli I, Hasannia S, Pirooznia N. A PLGA-encapsulated chimeric protein protects against adherence and toxicity of enterotoxigenic Escherichia coli. Microbiol Res 2014;169:205-212.
25. Jahantigh D, Saadati M, Fasihi Ramandi M, Mousavi M, Zand AM. Novel intranasal vaccine delivery system by chitosan nanofibrous membrane containing N-terminal region of Ipad antigen as a nasal shigellosis vaccine, studies in guinea pigs. J of Drug Delivery Sci Technol 2014; 24:33-39.
26. Bret D, Ulery BD, Kumar D, Ramer-Tait AE, Metzger DW, Wannemuehler MJ, et al. Design of a protective single-dose intranasal nanoparticle-based vaccine platform for respiratory infectious diseases. PLoS ONE. 2011; 6: e17642.
27. Nnamani PO, Scoles G, Kröl S. Preliminary characterization of N-trimethyl chitosan as a nanocarrier for malaria vaccine. J Vector Borne Dis 2011; 48:224-230.
28. Jadhav SS. Bhalerao AV. Formulation and characterization of chitosan nanoparticles loaded with rizatriptan benzoate. Der Pharmacia Lettre 2013; 5:218-223.
29. Bento D, Staats HF, Gonçalves T, Borges O. Development of a novel adjuvanted nasal vaccine: C48/80 associated with chitosan nanoparticles as a path to enhance mucosal immunity. Eur J Pharm Biopharm 2015; 93:143-164.
30. Biswas S, Chattopadhyay M, Sen KK, Saha MK. Development and characterization of alginate coated low molecular weight chitosan nanoparticles as new carriers for oral vaccine delivery in mice. Carbohydr Polym 2015;121:403-410.
31. Xu JH, Dai WJ, Chen B, Fan XY. Mucosal immunization with PsaA protein, using chitosan as a delivery system, increases protection against acute otitis media and invasive infection by Streptococcus pneumoniae. Scand J Immunol 2015;81:177-185.
32. Islam MA, Firdous J, Choi YJ, Yun CH, Cho CS. Design and application of chitosan microspheres as oral and nasal vaccine carriers: an updated review. Int J Nanomed 2012; 7:6077-6093.
33. Slütter B, Plapied L, Fievez V, Sande MA, des Rieux A, Schneider YJ, et al. Mechanistic study of the adjuvant effect of biodegradable nanoparticles in mucosal vaccination. J Controlled Release 2009; 138:113-121.
34. Van der Lubben IM, Kersten G, Fretz  MM, Beuvery C, Verhoef JC, Junginger HE. Chitosan microparticles for mucosal vaccination against diphtheria: oral and nasal efficacy studies in mice. Vaccine 2003; 21:1400-1408.
35. Vila A, Sa´nchez A, Janesa K, Behrensb I, Kisselb T, Vila Jato JL, et al. Low molecular weight chitosan nanoparticles as new carriers for nasal vaccine delivery in mice. Eur J Pharm Biopharm 2004; 57:123-131.
36. Rezaei Mokarram A, Alonso MJ. Preparation and evaluation of chitosan nanoparticles containing diphteria toxoid as new carriers for nasal vaccine delivery in mice. Arch Razi Inst 2006; 61:13-25.
37. Slütter B, Bala S, Keijzerb C, Mallantsc R, Hagenaarsd N, Que I. Nasal vaccination with N-trimethyl chitosan and PLGA based nanoparticles: Nanoparticle characteristics determine quality and strength of the antibody response in mice against the encapsulated antigen. Vaccine 2010; 28:6282-6291.
38. Siddhapura K, Harde H, Jain S. Immunostimulatory effect of tetanus toxoid loaded chitosan nanoparticles following microneedles assisted immunization. Nanomedicine 2016;12:213-222.
39. Kiyatkin N, Maksymowych AB, Simpson LL. Induction of an immune response by oral administration of recombinant botulinum toxin. Infect Immun 1997; 65:4586-4591.
40. Bagheripour MJ, Ebrahimi F, Hajizadeh A, Nazarian Sh. Immunogenicity effect of chitosan nanoparticles containing botulinum neurotoxin E binding domain recombinant protein in mice. J Mazandaran Univ Med Sci 2015; 25:37-47. (in Persian)
41. RabieeRudsari A, Ebrahimi F, ArefpourTorabi MA. Study of adjuvant capability of the gold nanoparticles on the immunity of botulinum neurotoxin serotype E in mouse. Sci J Adv Defence Sci Technol 2013; 4:87-92. (in Persian)