Original Article

Preparation of chitosan nanoparticle containing recombinant StxB antigen of EHEC and evaluation its immunogenicity in BALB/c mice


Background and Objectives: Escherichia coli O157:H7 is one of the most important food pathogens that produces colitis and bloody urine in humans. The Stx2B subunit is considered as one of the candidates for vaccine due to its immunogenic and adjuvant properties. Designing a mucosal vaccine using nanoparticles for protecting the antigen against degradation and controlling the release of antigen are important. The objective of the current study was to prepare nanoparticles containing the Stx2B subunit of E. coli O157:H7 and evaluation of its immunogenicity in the mouse model.
Materials and Methods: E. coli BL21 DE3 and pET28a-stxB were used for expression of the stx2b gene. After inducing gene expression, purification of the Stx2b protein was performed. Then, chitosan nanoparticle containing recombinant Stx2B was prepared and administered to BALB/c mice. IgA and IgG titers in serum and IgA titers in feces of immunized and control mice were evaluated by the ELISA method.
Results: After expression and purification of the Stx2B recombinant protein, an expected band of 13 kDa was observed on the SDS-PAGE gel and confirmed by Western Blot analysis. The size of the nanoparticle containing Stx2B was 290 nm. In the immunized mice, IgG and IgA titers were significantly increased. The immunized mice were challenged against E. coli O157:H7 Stx+ and the shedding analysis showed that colonization of bacteria in the intestinal tract decreased.
Conclusion: Oral administration of nanoparticles containing Stx2B as a candidate for the vaccine can induce a systemic and mucosal immune response against Stx2 toxin and can provide acceptable protection.

Nield BS, Holmes AJ, Gillings MR, Recchia GD, Mabbutt BC, Nevalainen K, et al. Recovery of new integron classes from environmental DNA. FEMS Microbiol Lett 2001; 195(1): 59-65.

Rowe-Magnus DA, Guerout A-M, Ploncard P, Dychinco B, Davies J, Mazel D. The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons. Proceedings of the National Academy of Sciences 2001; 98(2): 652-7.

Simoons-Smit A, Verwey-van Vught A, Kanis I, MacLaren D. Virulence of Klebsiella strains in experimentally induced skin lesions in the mouse. J Med Microbiol 1984; 17(1): 67-77.

Tängdén T, Cars O, Melhus Å, Löwdin E. Foreign travel is a major risk factor for colonization with Escherichia coli producing CTX-M-type extended-spectrum β-lactamases: a prospective study with Swedish volunteers. Antimicrob Agents Chemother 2010; 54(9): 3564-8.

Amidi M, Romeijn SG, Borchard G, Junginger HE, Hennink WE, Jiskoot W. Preparation and characterization of protein-loaded N-trimethyl chitosan nanoparticles as nasal delivery system. J Control Release 2006; 111(1): 107-16.

Sussman M. Escherichia coli: mechanisms of virulence: Cambridge University Press; 1997.

Johannes L, Römer W. Shiga toxins—from cell biology to biomedical applications. Nat Rev Microbiol 2010; 8(2): 105-16.

Obrig TG. Escherichia coli Shiga toxin mechanisms of action in renal disease. Toxins 2010; 2(12): 2769-94.

Sandvig K, Wälchli S, Lauvrak SU. Shiga toxins and their mechanisms of cell entry. Microbial Protein Toxins: Springer; 2004. p. 35-53.

Reddy ST, Van Der Vlies AJ, Simeoni E, Angeli V, Randolph GJ, O'Neil CP, et al. Exploiting lymphatic transport and complement activation in nanoparticle vaccines. Nat Biotechnol 2007; 25(10): 1159-64.

Felnerova D, Viret J-F, Glück R, Moser C. Liposomes and virosomes as delivery systems for antigens, nucleic acids and drugs. Curr Opin Biotechnol 2004; 15(6): 518-29.

Walker CLF, Aryee MJ, Boschi-Pinto C, Black RE. Estimating diarrhea mortality among young children in low and middle income countries. PLoS One 2012; 7(1): e29151.

Liu L, Johnson HL, Cousens S, Perin J, Scott S, Lawn JE, et al. Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. The Lancet 2012; 379(9832): 2151-61.

Jafari F, Shokrzadeh L, Hamidian M, Salmanzadeh-Ahrabi S, Zali MR. Acute diarrhea due to enteropathogenic bacteria in patients at hospitals in Tehran. Jpn J Infect Dis 2008; 61(4): 269-73.

te Loo DMW, Monnens LA, van der Velden TJ, Vermeer MA, Preyers F, Demacker PN, et al. Binding and transfer of verocytotoxin by polymorphonuclear leukocytes in hemolytic uremic syndrome. Blood 2000; 95(11): 3396-402.

Johnson RP, Clarke RC, Wilson JB, Read SC, Rahn K, Renwick SA, et al. Growing concerns and recent outbreaks involving non-O157: H7 serotypes of verotoxigenic Escherichia coli. Journal of Food Protection® 1996; 59(10): 1112-22.

Sabui S, Ghosal A, Dutta S, Ghosh A, Ramamurthy T, Nataro JP, et al. Allelic variation in colonization factor CS6 of enterotoxigenic Escherichia coli isolated from patients with acute diarrhoea and controls. J Med Microbiol 2010; 59(7): 770-9.

Stevens MP, Van Diemen PM, Dziva F, Jones PW, Wallis TS. Options for the control of enterohaemorrhagic Escherichia coli in ruminants. Microbiology 2002; 148(12): 3767-78.

Nazarian S, Gargari SLM, Rasooli I, Hasannia S, Pirooznia N. A PLGA-encapsulated chimeric protein protects against adherence and toxicity of enterotoxigenic Escherichia coli. Microbiol Res 2014; 169(2): 205-12.

Jiang Z-D, Mathewson JJ, Ericsson CD, Svennerholm A-M, Pulido C, DuPont HL. Characterization of Enterotoxigenic Eschevichia coli Strains in Patients with Travelers' Diarrhea Acquired in Guadalajara, Mexico, 1992–1997. J Infect Dis 2000; 181(2): 779-82.

Ferrari F, Bonferoni MC, Rossi S, Sandri G, Caramella CM. Manufacture Techniques of Chitosan‐Based Microparticles and Nanoparticles for Biopharmaceuticals. Chitosan-Based Systems for Biopharmaceuticals: Delivery, Targeting and Polymer Therapeutics 2007: 137-58.

Sable SB, Cheruvu M, Nandakumar S, Sharma S, Bandyopadhyay K, Kellar KL, et al. Cellular immune responses to nine Mycobacterium tuberculosis vaccine candidates following intranasal vaccination. PLoS One 2011; 6(7): e22718.

Sinha V, Trehan A. Biodegradable microspheres for protein delivery. J Control Release 2003; 90(3): 261-80.

Zhao K, Zhang Y, Zhang X, Shi C, Wang X, Wang X, et al. Chitosan-loaded poly (lactic-co-glycolic) acid nanoparticles as an efficient delivery system for Newcastle disease virus DNA vaccine. Int J Nanomedicine 2014; 9: 4609.

Pawar D, Goyal AK, Mangal S, Mishra N, Vaidya B, Tiwari S, et al. Evaluation of mucoadhesive PLGA microparticles for nasal immunization. The AAPS journal 2010; 12(2): 130-7.

Bowman K, Leong KW. Chitosan nanoparticles for oral drug and gene delivery. Int J Nanomedicine 2006; 1(2): 117.

IssueVol 10 No 6 (2018) QRcode
SectionOriginal Article(s)
Enterohemorrhagic Escherichia coli Stx2B subunit Nanoparticle chitosan Subunit vaccine

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How to Cite
Almasian P, Amani J, Baghban Arani F, Nazarian S, Kazemi R, Mirzaee Tabrizi N. Preparation of chitosan nanoparticle containing recombinant StxB antigen of EHEC and evaluation its immunogenicity in BALB/c mice. Iran J Microbiol. 2019;10(6):361-370.