Immunogenicity of enterotoxigenic Escherichia coli outer membrane vesicles encapsulated in chitosan nanoparticles

Document Type : Original Article


1 Department of Biology, Faculty of Science, Shahed University, Tehran, Iran

2 Department of Biology, Faculty of Science, Imam Hossein University, Tehran, Iran


Objective(s): Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease in humans, particularly in children under 5 years and travelers in developing countries.
To our knowledge, no vaccine is licensed yet to protect against ETEC infection. Like many Gram-negative pathogens, ETEC can secrete outer membrane vesicles (OMVs). These structures contain various immunogenic virulence proteins such as LT and therefore can be used as vaccine candidates. In this study we attempted to isolate the OMVs of ETEC cultivated at different temperatures and evaluate their immunogenicity and protective efficacy in a murine model of infection.
Materials and Methods: OMVs was purified from bacterial supernatant by ultracentrifugation. OMVs were encapsulated in chitosan nanoparticles prepared by ionic gelation method within a layer of Eudragit L100 for oral delivery.  Female BALB/c mice of 9 weeks’ old were immunized by parenteral injection and oral administration with free and encapsulated OMVs obtained from bacteria cultivated at 37°C and 42°C. The serum samples were collected and the antibody titers were measured by an enzyme-linked immunosorbent assay (ELISA).
Results: The protein concentrations of OMVs were 3.47 mg/ml and 2.46 mg/ml for bacteria grown at 37°C and 42°C respectively. OMVs loaded into nanoparticles (NP-OMVs) were homogeneous and spherical in shape, with a size of 532 nm. The encapsulation efficiency of NP was 90%. Mice immunized with OMVs, inhibited the ETEC colonization in their small intestine and induced production of antibodies against LT toxin.
Conclusion: The results obtained in this research place OMVs among promising candidates to be used for vaccination.


Main Subjects

1. Nazarian S, Gargari SL, Rasooli I, Alerasol M, Bagheri S, Alipoor SD. Prevalent phenotypic and genotypic profile of enterotoxigenic Escherichia coli among Iranian children. Jpn J Infect Dis 2014;67:78-85.
2. Qadri F, Svennerholm AM, Faruque ASG, Sack RB. Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev2005;18:465-483.
3. Nazarian S, Mousavi Gargari SL, Rasooli I, Amani J, Bagheri S, Alerasool M. An in silico chimeric multi subunit vaccine targeting virulence factors of enterotoxigenic Escherichia coli (ETEC) with its bacterial inbuilt adjuvant. J Microbiol Methods2012;90:36-45.
4. Jiang ZD, Lowe B, Verenkar MP, Ashley D, Steffen R, Tornieporth N, et al. Prevalence of enteric pathogens among international travelers with diarrhea acquired in Kenya (Mombasa), India (Goa), or Jamaica (Montego Bay).J Infect Dis2002;185:497-502
5. Katz DE, DeLorimier AJ, Wolf MK, Hall ER, Cassels FJ, van Hamont  JE,et al. Oral immunization of adult volunteers with microencapsulated enterotoxigenic Escherichia coli (ETEC) CS6 antigen. Vaccine 2003;21:341-346.
6. Steffen R, Castelli F, Dieter Nothdurft H, Rombo L, Jane Zuckerman N,et al. Vaccination against Enterotoxigenic Escherichia coli, a Cause of Travelers Diarrhea. J Travel Med2005;12:102-107.
7. Nazarian S, Gargari SL, Rasooli I, Hasannia S, Pirooznia N,et al. A PLGA-encapsulated chimeric protein protects against adherence and toxicity of enterotoxigenic Escherichia coli. Microbiol Res 2014;169:205-212.
8. Walker RI, Steele D, Aguado T, Committee AHETE. Analysis of strategies to successfully vaccinate infants in developing countries against enterotoxigenic E. coli (ETEC) disease. Vaccine 2007;25:2545-2566.
9. Lundgren A, Bourgeois L, Carlin N, Clements J, Gustafsson B, Hartford M, et al. Safety and immunogenicity of an improved oral inactivated multivalent enterotoxigenic Escherichia coli (ETEC) vaccine administered alone and together with dmLT adjuvant in a double-blind, randomized, placebo-controlled phase I study. Vaccine2014;32:7077-7084.
10. Kulp A, Kuehn MJ. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu Review Microbiol.2010;64:163-184.
11. Roy K, Bartels S, Qadri F, Fleckenstein JM. Enterotoxigenic Escherichia coli elicits immune responses to multiple surface proteins. Infect Immun.2010;78:3027-3035.
12. Roy K, Hamilton DJ, Munson GP, Fleckenstein JM ,et al. Outer membrane vesicles induce immune responses to virulence proteins and protect against colonization by enterotoxigenic Escherichia coli. Clin Vaccine immunol.2011;18:1803-1808.
13. Bishop AL, Schild S, Patimalla B, Klein B, Camilli A. Mucosal immunization with Vibrio cholerae outer membrane vesicles provides maternal protection mediated by antilipopolysaccharide antibodies that inhibit bacterial motility. Infect Immun.2010;78:4402-4420.
14. Holst J, Martin D, Arnold R, Huergo  CC, Oster  P, O´Hallahan J,et al. Properties and clinical performance of vaccines containing outer membrane vesicles from Neisseria meningitidis. Vaccine2009;27:B3-B12.
15. Keenan JI, Rijpkema SG, Durrani Z, Roake JA. Differences in immunogenicity and protection in mice and guinea pigs following intranasal immunization with Helicobacter pylori outer membrane antigens. FEMS Immunol Med Microbiol2003;36:199-205.
16. Acevedo R, Fernandez S, Zayas C, Acosta A, Sarmiento ME, Ferro VA,et al. Bacterial outer membrane vesicles and vaccine applications. Front Immunol 2014;25.121.
17. Danzig L. Meningococcal vaccines.PediatrInfect Dis J2004;23:S285-S292.
18. Leitner DR, Zingl FG, Schild S. A glimpse on outer membrane vesicles as vaccine candidates.  Vaccines Vaccin 2015;6:293.
19. Leitner DR, Lichtenegger S, Temel P, Zingl FG, Ratzberger D, Roier S,et al. A combined vaccine approach against Vibrio cholerae and ETEC based on outer membrane vesicles. Front Microbiol 2015;6:823.
20. Sanchez J, olmgren J. Virulence factors, pathogenesis and vaccine protection in cholera and ETEC diarrhea. Curr Opin Immunol2005;17:388-398.
21. Kulkarni HM, Jagannadham MV. Biogenesis and multifaceted roles of outer membrane vesicles from Gram-negative bacteria. Microbiology2014;160:2109-2121.
22. MacDonald IA, Kuehn MJ. Stress-induced outer membrane vesicle production by Pseudomonas aeruginosa. J Bacteriol2013;195:2971-2981.
23. Aziz MA, Midha S, Waheed SM, Bhatnagar  R. Oral vaccines: new needs, new possibilities. Bioessays 2007;29:591-604.
24. Wang S, Liu H, Zhang X, Qian  F. Intranasal and oral vaccination with protein-based antigens: advantages, challenges and formulation strategies. Protein Cell2015;6:480-503.
25. Koide SS. Chitin-chitosan: properties, benefits and risks. Nutr Res1998;18:1091-1101.
26. Ensign LM, Cone R, Hanes J. Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers. Adv Drug Deliv Rev2012;64:557-570.
27. Badhana S, Garud N, Garud A. Colon specific drug delivery of mesalamine using eudragit S100-coated chitosan microspheres for the treatment of ulcerative colitis. Int Curr Pharm J2013;2:42-48.
28. Delbaz SA, Siadat SD, Aghasadeghi MR, Piryaie M, Peerayeh SN, Mousavi SF, et al. Biological and immunological evaluation of Neisseria meningitidisserogroup A outer membrane vesicle as vaccine candidates. Jundishapur J Microbiol2013;6.e5007.
29. Sawasvirojwong S, Srimanote P, Chatsudthipong V, Muanprasat  C, et al. An adult mouse model of Vibrio cholerae-induced diarrhea for studying pathogenesis and potential therapy of Cholera. PLoS Negl Trop Dis2013;7:e2293.
30. Alerasol M, Mousavi Gargari SL, Nazarian S, Bagheri  S, et al. Immunogenicity of a fusion protein comprising coli surface antigen 3 and labile B subunit of enterotoxigenic Escherichia coli. Iran Biomed J 2014;18:212-218.
31. Bagheri S, Mousavi Gargari SL, Rasooli I, Nazarian S, Alerasol M. A CssA, CssB and LTB chimeric protein induces protection against Enterotoxigenic Escherichia coli. Braz J Infect Dis 2014;18:308-314.
32. Roy K, Hamilton D, Allen KP, Randolph MP, Fleckenstein JM, et al. The EtpA exoprotein of enterotoxigenic Escherichia coli promotes intestinal colonization and is aprotec tive
antigen in an experimental model of murine infection. Infect Immun2008;76:2106-2112.
33. Callico A, Fernandez S, Cabrera R, Acosta M, Aranguren Y, Fernandez Y. Proteoliposomes derived from Escherichia coil induced systemic and mucosal response. VacciMonitor2010;19.32.
34. Schild S, Nelson EJ, Camilli A. Immunization with Vibrio cholerae outer membrane vesicles induces protective immunity in mice. Infect Immun2008;76:4554-4563.
35. Norheim G, Tunheim G, Naess LM, Kristiansen  PA, Caugant  DA, Rosenqvist E. An Outer Membrane Vesicle Vaccine for Prevention of Serogroup A and W_135 Meningococcal Disease in the African Meningitis Belt. Scand J Immunol2012;76:99-107.
36. Van de Waterbeemd B, Streefland M, Van der Ley P, Zomer B, Van Dijken H, Martens D. Improved OMV vaccine against Neisseria meningitidis using genetically engineered strains and a detergent-free purification process. Vaccine2010;28:4810-4816.
37. Klimentova J, Stulik J. Methods of isolation and purification of outer membrane vesicles from gram-negative bacteria. Microbiol Res2015;170:1-9.
38. Davitt CJ, Lavelle EC. Delivery strategies to enhance oral vaccination against enteric infections. Adv Drug Deliv Rev2015;91:52-69.
39. Lee WJ, Cha S, Shin M, Jung M, Islam MA, Cho CS. Efficacy of thiolated eudragit microspheres as an oral vaccine delivery system to induce mucosal immunity against enterotoxigenic Escherichia coli in mice. Eur JPharm Biopharm2012;81:43-48.
40. Camacho AI, Irache JM, de SJ, Sanchez-Gomez  S, Gamazo C. Nanoparticle-based vaccine for mucosal protection against Shigella flexneri in mice. Vaccine2013;31:3288-3294.
41. Elgadir MA, Uddin M, Ferdosh S, Adam  A, Chowdhury  AJK, Sarker M. Impact of chitosan composites and chitosan nanoparticle composites on various drug delivery systems: A review.  J Food Drug Anal2015; 23.619-629.