Immunogenicity of inactivated Escherichia coli O157:H7 with Stx2B microparticle in mice

Document Type : Original Article


1 Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran

2 Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

3 Department of Biology, Imam Hussein University, Tehran, Iran



Objective(s): Vaccination using inactivated bacteria is one of the most effective ways to protect against EHEC infection. Escherichia coli O157:H7 infections are mainly influenced by Shiga toxins (Stx) and attaching/effacing factors. Among various factors, Stx2B is gaining much attention as a vaccine candidate. Formulating an inactivated bacteria with a suitable adjuvant increases vaccine efficacy and antibody production and can lead to a lasting immune response and protection against O157:H7. 
Materials and Methods: To assess vaccine efficacy, in this study, we have considered heat and formalin-inactivated bacteria along with chitosan-coated Stx2B/ Stx2B in a mouse model. Ionotropic gelation via tripolyphosphate anions was used to coat Stx2B on chitosan. Subcutaneous injection or oral gavage was used to immunize mice, which were then challenged with E. coli O157:H7. 
Results: Immunity and protection against E. coli  O157:H7 were achieved by all forms of the vaccine. Inactivated E. coli  O157:H7 formulated with chitosan-coated Stx2B effectively evoked humoral and mucosal immune responses. However, minimum shedding appeared with the mice groups orally immunized with formalin-inactivated bacteria sublimated with chitosan-coated Stx2B and heat-inactivated bacteria plus Stx2B in subcutaneous immunization.
Conclusion: Administration of inactivated whole-cell and toxin was synergistic and increased the protection capacity with both parenteral and oral immunization routes.


1. Tarazi YH, El-Sukhon SN, Ismail ZB, Almestarehieh AA. Molecular characterization of enterohemorrhagic Escherichia coli isolated from diarrhea samples from human, livestock, and ground beef in north jordan. Vet World 2021; 14:2827-2832.
2. Heredia N, García S. Animals as sources of food-borne pathogens: a review. Anim Nutr 2018; 4:250-255.
3. Álvarez RS, Gómez FD, Zotta E, Paton AW, Paton JC, Ibarra C, et al. Combined action of shiga toxin type 2 and subtilase cytotoxin in the pathogenesis of hemolytic uremic syndrome. Toxins 2021; 13:536-552.
4. Smith JL, Fratamico PM. Emerging and re-emerging foodborne pathogens. Foodborne Pathog Dis 2018; 15:737-757.
5. Ameer MA, Wasey A, Salen P. Escherichia coli (E. coli 0157 H7).  StatPearls. Treasure Island (FL): StatPearls publishing copyright © 2021, StatPearls Publishing LLC.; 2021.
6. Menge C. The role of Escherichia coli Shiga toxins in STEC colonization of cattle. Toxins 2020; 12:607-644.
7. Sanchez-Villamil JI, Tapia D, Torres AG. Development of a gold nanoparticle vaccine against enterohemorrhagic Escherichia coli O157: H7. MBio 2019; 10:1869-1819.
8. Segura A, Bertin Y, Durand A, Benbakkar M, Forano E. Transcriptional analysis reveals specific niche factors and response to environmental stresses of enterohemorrhagic Escherichia coli O157: H7 in bovine digestive contents. BMC Microbiol 2021; 21:1-16.
9. Mohawk KL, Melton-Celsa AR, Robinson CM, O’Brien AD. Neutralizing antibodies to Shiga toxin type 2 (Stx2) reduce colonization of mice by Stx2-expressing Escherichia coli O157: H7. Vaccine 2010; 28:4777-4785.
10. Yang B, Jiang L, Wang S, Wang L. Global transcriptional regulation by BirA in enterohemorrhagic Escherichia coli O157: H7. Future Microbiol 2018; 13:757-769.
11. Węgrzyn G, Muniesa M. Shiga toxin-converting bacteriophages. Front Microbiol 2021; 12:1-3.
12. Lawan A, Jesse F, Idris U, Odhah M, Arsalan M, Muhammad N, et al. Mucosal and systemic responses of immunogenic vaccines candidates against enteric Escherichia coli infections in ruminants: a review. Microb Pathog 2018; 117:175-183.
13. Ansari A, Madan A, Prakash D. Vaccine development—a complex science. EPRA Int J Multidiscip Res 2021; 7:34-37.
14. Parhi R. Drug delivery applications of chitin and chitosan: a review. Environ Chem Lett 2020; 18:577-594.
15. Jazayeri SD, Lim HX, Shameli K, Yeap SK, Poh CL. Nano and microparticles as potential oral vaccine carriers and adjuvants against infectious diseases. Front Pharmacol 2021; 12:1399-1414.
16. Vimal S, Majeed SA, Taju G, Nambi K, Raj NS, Madan N, et al. RETRACTED: chitosan tripolyphosphate (CS/TPP) nanoparticles: preparation, characterization and application for gene delivery in shrimp. Acta Tropica 2013; 128:486-493.
17. Marsili L, Dal Bo M, Berti F, Toffoli G. Thermoresponsive chitosan-grafted-Poly (N-Vinylcaprolactam) microgels via ionotropic gelation for oncological applications. Pharmaceutics 2021; 13:1654-1680.
18. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: cold spring harbor laboratory press New York; 1989.
19. Kazemi R, Akahavian Tehrani A, Jafari M, Amani J, Mousavi A, Salmanian AH. Cloning and expression of the binding subunit of shiga-like toxin type 2 gene and immunization study in an animal model. Pathobiol Res 2016; 18:45-60.
20. Arshadi N, Mousavi SL, Amani J, Nazarian S. Immunogenic potency of formalin and heat inactivated E. coli O157: H7 in mouse model administered by different routes. Avicenna J Med Biotechnol 2020; 12:194-200.
21. Stoica R, Şomoghi R, Ion R. Preparation of chitosan-tripolyphosphate nanoparticles for the encapsulation of polyphenols extracted from rose hips. Dig J Nanomater Biostructures 2013; 8:955-963.
22. Safer AM, Leporatti S, Jose J, Soliman MS. Conjugation of EGCG and chitosan NPs as a novel nano-drug delivery system.  Int J Nanomedicine 2019; 14:8033-8046.
23. Farkas N, Kramar JA. Dynamic light scattering distributions by any means. J Nanoparticle Res 2021; 23:1-11.
24. Hassani S, Laouini A, Fessi H, Charcosset C. Preparation of chitosan–TPP nanoparticles using microengineered membranes–effect of parameters and encapsulation of tacrine. Colloids  Surf A: Physicochem Eng Asp 2015; 482:34-43.
25. Khanifar J, Salmanian AH, Haji Hosseini R, Amani J, Kazemi R. Chitosan nano-structure loaded with recombinant Escherichia coli O157:H7 antigens as a vaccine candidate can effectively increase immunization capacity. Artif Cells Nanomed Biotechnol 2019; 47:2593-2604.
26. Yu S, Gu J, Wang H-g, Wang Q-x, Luo P, Wu C, et al. Identification of a novel linear epitope on EspA from enterohemorrhagic Escherichia coli using a neutralizing and protective monoclonal antibody. Clin Immunol 2011; 138:77-84.
27. Bedasa S, Shiferaw D, Abraha A, Moges T. Occurrence and antimicrobial susceptibility profile of Escherichia coli O157:H7 from food of animal origin in Bishoftu town, central ethiopia. Int J Food Contam 2018; 5:2-9.
28. Mühlen S, Dersch P. Treatment strategies for infections with shiga toxin-producing Escherichia coli. Front Cell Infect Microbiol 2020; 10:169-186.
29. Martorelli L, Garbaccio S, Vilte DA, Albanese AA, Mejías MP, Palermo MS, et al. Immune response in calves vaccinated with type three secretion system antigens and shiga toxin 2b subunit of Escherichia coli O157: H7. PLoS One 2017; 12:169422-169437.
30. Albanese A, Sacerdoti F, Seyahian EA, Amaral MM, Fiorentino G, Fernandez Brando R, et al. Immunization of pregnant cows with Shiga toxin-2 induces high levels of specific colostral antibodies and lactoferrin able to neutralize E. coli O157:H7 pathogenicity. Vaccine 2018; 36:1728-1735.
31. Mwirigi M, Nkando I, Aye R, Soi R, Ochanda H, Berberov E, et al. Experimental evaluation of inactivated and live attenuated vaccines against Mycoplasma mycoides subsp. mycoides. Vet Immunol Immunopathol 2016; 169:63-67.
32. Mejias MP, Ghersi G, Craig PO, Panek CA, Bentancor LV, Baschkier A, et al. Immunization with a chimera consisting of the B subunit of Shiga toxin type 2 and brucella lumazine synthase confers total protection against Shiga toxins in mice. J Immunol 2013; 191:2403-2411.
33. Tartour E, Johannes L. STxB as an antigen delivery tool for mucosal maccination. Toxins 2022; 14:202-222.
34. Li X, Xing R, Xu C, Liu S, Qin Y, Li K, et al. Immunostimulatory effect of chitosan and quaternary chitosan: a review of potential vaccine adjuvants. Carbohydr Polym 2021; 264:118050-118066.