Medical Microbiology and Immunology

, Volume 208, Issue 1, pp 89–100 | Cite as

Intranasal co-administration of recombinant active fragment of Zonula occludens toxin and truncated recombinant EspB triggers potent systemic, mucosal immune responses and reduces span of E. coli O157:H7 fecal shedding in BALB/c mice

  • Aravind Shekar
  • Shylaja Ramlal
  • Joseph Kingston Jeyabalaji
  • Murali Harishchandra SripathyEmail author
Original Investigation


Escherichia coli O157:H7 with its traits such as intestinal colonization and fecal-oral route of transmission demands mucosal vaccine development. E. coli secreted protein B (EspB) is one of the key type III secretory system (TTSS) targets for mucosal candidate vaccine due to its indispensable role in the pathogenesis of E. coli O157:H7. However, mucosally administered recombinant proteins have low immunogenicity which could be overcome by the use of mucosal adjuvants. The quest for safe, potent mucosal adjuvant has recognized ΔG fragment of Zonula occludens toxin of Vibrio cholerae with such properties. ΔG enhances mucosal permeability via the paracellular route by altering epithelial tight junction structure in a reversible, ephemeral and non-toxic manner. Therefore, we tested whether recombinant ΔG intranasally co-administered with truncated EspB (EspB + ΔG) could serve as an effective mucosal adjuvant. Results showed that EspB + ΔG group induced higher systemic IgG and mucosal IgA than EspB alone. Moreover, EspB alone developed Th2 type response with IgG1/IgG2a ratio (1.64) and IL-4, IL-10 cytokines whereas that of EspB + ΔG group generated mixed Th1/Th2 type immune response evident from IgG1/IgG2a ratio (1.17) as well as IL-4, IL-10 and IFN-γ cytokine levels compared to control. Sera of EspB + ΔG group inhibited TTSS mediated haemolysis of murine RBCs more effectively compared to EspB, control group and sera of both EspB + ΔG, EspB group resulted in similar levels of efficacious reduction in E. coli O157:H7 adherence to Caco-2 cells compared to control. Moreover, vaccination with EspB + ΔG resulted in significant reduction in E. coli O157:H7 fecal shedding compared to EspB and control group in experimentally challenged streptomycin-treated mice. These results demonstrate mucosal adjuvanticity of ΔG co-administered with EspB in enhancing overall immunogenicity to reduce E. coli O157:H7 shedding.


E. coli O157 Ruminants Immunization Intranasal administration Feces Vaccines 



Aravind S is funded by Senior Research Fellowship from Defence Research and Development Organisation, India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that no conflict of interest exists.

Ethical approval

For this type of study, formal consent is not required.

Statement on the welfare of animals

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution as per the Institutional Animal Ethical Committee (IAEC code 15/2016), DFRL, Mysuru, Karnataka, India.

Data availability statement

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


  1. 1.
    Karmali MA, Gannon V, Sargeant JM (2010) Verocytotoxin-producing Escherichia coli (VTEC). Vet Microbiol 140:360–370CrossRefGoogle Scholar
  2. 2.
    Varela NP, Dick P, Wilson J (2013) Assessing the existing information on the efficacy of bovine vaccination against Escherichia coli o157: H7—a systematic review and meta-analysis. Zoonoses Public Health 60:253–268CrossRefGoogle Scholar
  3. 3.
    Matthews L, Reeve R, Gally DL et al (2013) Predicting the public health benefit of vaccinating cattle against Escherichia coli O157. Proc Natl Acad Sci USA 110:16265–16270. CrossRefGoogle Scholar
  4. 4.
    Potter AA, Klashinsky S, Li Y et al (2004) Decreased shedding of Escherichia coli O157:H7 by cattle following vaccination with type III secreted proteins. Vaccine 22:362–369. CrossRefGoogle Scholar
  5. 5.
    McNeilly TN, Mitchell MC, Rosser T et al (2010) Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157:H7 following oral challenge. Vaccine 28:1422–1428. CrossRefGoogle Scholar
  6. 6.
    Lin R, Zhu B, Zhang Y et al (2017) Intranasal immunization with novel EspA-Tir-M fusion protein induces protective immunity against enterohemorrhagic Escherichia coli O157:H7 challenge in mice. Microb Pathog 105:19–24. CrossRefGoogle Scholar
  7. 7.
    Wan C, Zhou Y, Yu Y et al (2011) B-cell epitope KT-12 of enterohemorrhagic Escherichia coli O157:H7: a novel peptide vaccine candidate. Microbiol Immunol 55:247–253. CrossRefGoogle Scholar
  8. 8.
    Nart P, Holden N, McAteer SP et al (2008) Mucosal antibody responses of colonized cattle to Escherichia coli O157-secreted proteins, flagellin, outer membrane proteins and lipopolysaccharide. FEMS Immunol Med Microbiol 52:59–68. CrossRefGoogle Scholar
  9. 9.
    Fasano A, Baudry B, Pumplin DW et al (1991) Vibrio cholerae produces a second enterotoxin, which affects intestinal tight junctions. Proc Natl Acad Sci USA 88:5242–5246. CrossRefGoogle Scholar
  10. 10.
    Fasano A, Uzzau S (1997) Modulation of intestinal tight junctions by zonula occludens toxin permits enteral administration of insulin and other macromolecules in an animal model. J Clin Invest 99:1158–1164. CrossRefGoogle Scholar
  11. 11.
    Fasano A, Fiorentini C, Donelli G et al (1995) Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro. J Clin Invest 96:710–720. CrossRefGoogle Scholar
  12. 12.
    Marinaro M, Fasano A, De Magistris MT (2003) Zonula occludens toxin acts as an adjuvant through different mucosal routes and induces protective immune responses. Infect Immun 71:1897–1902. CrossRefGoogle Scholar
  13. 13.
    Di Pierro M, Lu R, Uzzau S et al (2001) Zonula occludens toxin structure-function analysis. Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain. J Biol Chem 276:19160–19165. CrossRefGoogle Scholar
  14. 14.
    Salama NN, Fasano A, Thakar M, Eddington ND (2005) The impact of ∆G on the oral bioavailability of low bioavailable therapeutic agents. J Pharmacol Exp Ther 312:199–205. CrossRefGoogle Scholar
  15. 15.
    Pavot V, Rochereau N, Genin C et al (2012) New insights in mucosal vaccine development. Vaccine 30:142–154. CrossRefGoogle Scholar
  16. 16.
    Hamada D, Hamaguchi M, Suzuki KN et al (2010) Cytoskeleton-modulating effectors of enteropathogenic and enterohemorrhagic Escherichia coli: a case for EspB as an intrinsically less-ordered effector. FEBS J 277:2409–2415. CrossRefGoogle Scholar
  17. 17.
    Frankel G, Phillips AD, Rosenshine I et al (1998) Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements. Mol Microbiol 30:911–921. CrossRefGoogle Scholar
  18. 18.
    Kenny B, Finlay BB (1995) Protein secretion by enteropathogenic Escherichia coli is essential for transducing signals to epithelial cells. Proc Natl Acad Sci USA 92:7991–7995CrossRefGoogle Scholar
  19. 19.
    Asper DJ, Karmali MA, Townsend H et al (2011) Serological response of Shiga toxin-producing Escherichia coli type III secreted proteins in sera from vaccinated rabbits, naturally infected cattle, and humans. Clin Vaccine Immunol 18:1052–1057. CrossRefGoogle Scholar
  20. 20.
    Reddy PK, Ramlal S, Sripathy MH, Batra H (2012) A simple and universal ligation mediated fusion of genes based on hetero-staggered PCR for generating immunodominant chimeric proteins. Gene 509:104–109. CrossRefGoogle Scholar
  21. 21.
    Reichelt P, Schwarz C, Donzeau M (2006) Single step protocol to purify recombinant proteins with low endotoxin contents. Protein Expr Purif 46:483–488. CrossRefGoogle Scholar
  22. 22.
    Tadepalli G, Konduru B, Murali HS, Batra HV (2017) Intraperitoneal administration of a novel chimeric immunogen (rOP) elicits IFN-γ and IL-12p70 protective immune response in BALB/c mice against virulent Brucella. Immunol Lett 192:79–87. CrossRefGoogle Scholar
  23. 23.
    Amani J, Mousavi SL, Rafati S, Salmanian AH (2011) Immunogenicity of a plant-derived edible chimeric EspA, Intimin and Tir of Escherichia coli O157:H7 in mice. Plant Sci 180:620–627. CrossRefGoogle Scholar
  24. 24.
    Reddy PN, Paul S, Sripathy MH, Batra HV (2015) Evaluation of recombinant SEA-TSST fusion toxoid for protection against superantigen induced toxicity in mouse model. Toxicon 103:106–113. CrossRefGoogle Scholar
  25. 25.
    Martorelli L, Garbaccio S, Vilte DA et al (2017) Immune response in calves vaccinated with type three secretion system antigens and shiga toxin 2b subunit of Escherichia coli O157:H7. PLoS One 12:e0169422. CrossRefGoogle Scholar
  26. 26.
    Ruan X, Sack DA, Zhang W (2015) Genetic fusions of a CFA/I/II/IV MEFA (multiepitope fusion antigen) and a toxoid fusion of heat-stable toxin (STa) and heat-labile toxin (LT) of enterotoxigenic Escherichia coli (ETEC) retain broad anti-CFA and antitoxin antigenicity. PLoS One 10:e0121623. CrossRefGoogle Scholar
  27. 27.
    Amani J, Salmanian AH, Rafati S, Mousavi SL (2010) Immunogenic properties of chimeric protein from espA, eae and tir genes of Escherichia coli O157:H7. Vaccine 28:6923–6929. CrossRefGoogle Scholar
  28. 28.
    Snedeker KG, Campbell M, Sargeant JM (2012) A systematic review of vaccinations to reduce the shedding of Escherichia coli O157 in the faeces of domestic ruminants. Zoonoses Public Health 59:126–138. CrossRefGoogle Scholar
  29. 29.
    Yekta MA, Goddeeris BM, Vanrompay D, Cox E (2011) Immunization of sheep with a combination of intiminγ, EspA and EspB decreases Escherichia coli O157:H7 shedding. Vet Immunol Immunopathol 140:42–46. CrossRefGoogle Scholar
  30. 30.
    Vilte DA, Larzábal M, Garbaccio S et al (2011) Reduced faecal shedding of Escherichia coli O157:H7 in cattle following systemic vaccination with γ-intimin C280 and EspB proteins. Vaccine 29:3962–3968. CrossRefGoogle Scholar
  31. 31.
    Cataldi A, Yevsa T, Vilte DA et al (2008) Efficient immune responses against Intimin and EspB of enterohaemorragic Escherichia coli after intranasal vaccination using the TLR2/6 agonist MALP-2 as adjuvant. Vaccine 26:5662–5667. CrossRefGoogle Scholar
  32. 32.
    Ahmed B, Loos M, Vanrompay D, Cox E (2014) Oral immunization with Lactococcus lactis-expressing EspB induces protective immune responses against Escherichia coli O157:H7 in a murine model of colonization. Vaccine 32:3909–3916. CrossRefGoogle Scholar
  33. 33.
    Lin R, Zhang Y, Long B et al (2017) Oral immunization with recombinant lactobacillus acidophilus expressing espA-Tir-M confers protection against enterohemorrhagic Escherichia coli O157:H7 challenge in mice. Front Microbiol 8:417. Google Scholar
  34. 34.
    Chiu H-J, Syu W-J (2005) Functional analysis of EspB from enterohaemorrhagic Escherichia coli. Microbiology 151:3277–3286. CrossRefGoogle Scholar
  35. 35.
    Baumann D, Salia H, Greune L et al (2018) Multitalented EspB of enteropathogenic Escherichia coli (EPEC) enters cells autonomously and induces programmed cell death in human monocytic THP-1 cells. Int J Med Microbiol 308:387–404. CrossRefGoogle Scholar
  36. 36.
    Viswanathan VK, Koutsouris A, Lukic S et al (2004) Comparative analysis of EspF from enteropathogenic and enterohemorrhagic Escherichia coli in alteration of epithelial barrier function. Infect Immun 72:3218–3227. CrossRefGoogle Scholar
  37. 37.
    Tezuka H, Abe Y, Asano J et al (2011) Prominent role for plasmacytoid dendritic cells in mucosal T cell-independent IgA induction. Immunity 34:247–257. CrossRefGoogle Scholar
  38. 38.
    Ruane D, Brane L, Reis BS et al (2013) Lung dendritic cells induce migration of protective T cells to the gastrointestinal tract. J Exp Med 210:1871–1888. CrossRefGoogle Scholar
  39. 39.
    Yamamoto M, Vancott JL, Okahashi N et al (1996) The role of Th1 and Th2 cells for mucosal IgA responses. Ann N Y Acad Sci 778:64–71. CrossRefGoogle Scholar
  40. 40.
    Sollid LM, Kvale D, Brandtzaeg P et al (1987) Interferon-gamma enhances expression of secretory component, the epithelial receptor for polymeric immunoglobulins. J Immunol (Baltimore Md 1950) 138:4303–4306Google Scholar
  41. 41.
    Rüter C, Lubos ML, Norkowski S, Schmidt MA (2018) All in—Multiple parallel strategies for intracellular delivery by bacterial pathogens. Int J Med Microbiol. Google Scholar
  42. 42.
    Etcheverría AI, Padola NL (2013) Shiga toxin-producing Escherichia coli: factors involved in virulence and cattle colonization. Virulence 4:366–372CrossRefGoogle Scholar
  43. 43.
    Martorelli L, Garimano N, Fiorentino GA et al (2018) Efficacy of a recombinant Intimin, EspB and Shiga toxin 2B vaccine in calves experimentally challenged with Escherichia coli O157:H7. Vaccine 36:3949–3959CrossRefGoogle Scholar
  44. 44.
    García-Angulo VA, Kalita A, Kalita M et al (2014) Comparative genomics and immunoinformatics approach for the identification of vaccine candidates for enterohemorrhagic Escherichia coli O157:H7. Infect Immun 82:2016–2026. CrossRefGoogle Scholar
  45. 45.
    Babiuk S, Asper DJ, Rogan D et al (2008) Subcutaneous and intranasal immunization with type III secreted proteins can prevent colonization and shedding of Escherichia coli O157:H7 in mice. Microb Pathog 45:7–11. CrossRefGoogle Scholar
  46. 46.
    Yoshida M, Claypool SM, Wagner JS et al (2004) Human neonatal Fc receptor mediates transport of IgG into luminal secretions for delivery of antigens to mucosal dendritic cells. Immunity 20:769–783. CrossRefGoogle Scholar
  47. 47.
    Fan H, Wang L, Luo J, Long B (2012) Protection against Escherichia coli O157:H7 challenge by immunization of mice with purified Tir proteins. Mol Biol Rep 39:989–997. CrossRefGoogle Scholar
  48. 48.
    Mantis NJ, Rol N, Corthésy B (2011) Secretory IgA’s complex roles in immunity and mucosal homeostasis in the gut. Mucosal Immunol 4:603–611. CrossRefGoogle Scholar
  49. 49.
    Gould LH, Mody RK, Ong KL et al (2013) Increased recognition of non-O157 shiga toxin–producing Escherichia coli infections in the United States during 2000–2010: epidemiologic features and comparison with E. coli O157 Infections. Foodborne Pathog Dis 10:453–460. CrossRefGoogle Scholar
  50. 50.
    Brooks JT, Sowers EG, Wells JG et al (2005) Non-O157 shiga toxin–producing Escherichia coli infections in the United States, 1983–2002. J Infect Dis 192:1422–1429. CrossRefGoogle Scholar
  51. 51.
    McNeilly TN, Mitchell MC, Corbishley A et al (2015) Optimizing the protection of cattle against Escherichia coli O157:H7 colonization through immunization with different combinations of H7 Flagellin, Tir, Intimin-531 or EspA. PLoS One 10:e0128391. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Aravind Shekar
    • 1
  • Shylaja Ramlal
    • 1
  • Joseph Kingston Jeyabalaji
    • 1
  • Murali Harishchandra Sripathy
    • 1
    • 2
    Email author
  1. 1.Department of MicrobiologyDefence Food Research LaboratoryMysuruIndia
  2. 2.MysuruIndia

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