An LTB-entrapped protein in PLGA nanoparticles preserves against enterotoxin of enterotoxigenic Escherichia coli

Document Type : Original Article

Authors

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

Abstract

Objective(s): Enterotoxigenic Escherichia coli (ETEC) is known as the most common bacterial causes of diarrheal diseases related to morbidity and mortality. Heat-labile enterotoxin (LT) is a part of major virulence factors in ETEC pathogenesis. Antigen entrapment into nanoparticles (NPs) can protect them and enhance their immunogenicity.
Materials and Methods: In the present study, recombinant LTB protein was expressed in E. coli BL21 (DE3) and purified by an Ni-NTA agarose column. The protein was entrapped in PLGA polymer by the double emulsion method. NPs were characterized physicochemically and the protein release from the NPs was evaluated. ELISA assay was performed for investigation of raised antibody against the recombinant protein in mice. The anti-toxicity and anti-adherence attributes of the immune sera against ETEC were also evaluated.
Results: It showed the successful cloning of a 313 bp DNA fragment encoding LTB protein in the pET28a vector. Over-expression in BL21 (DE3) led to the formation of corresponding 15.5 kDa protein bands in the SDS-PAGE gel. Western blotting by using anti-CTX confirmed the purified LTB. Protein-entrapped NPs had a spherical shape with the size of 238 nm mean diameter and 85% entrapment efficiency.  Immunological analyses showed the production of a high titer of specific IgG antibody in immunized animals. The neutralizing antibody in the sera of immunized animals was approved by GM1 binding and Ileal loop assays.
Conclusion: The results indicate the efficacy of the entrapped LTB protein as an effective immunogen which induces the humoral responses.

Keywords

Main Subjects

References

1. Mirhoseini A, Amani J, Nazarian S. Review on pathogenicity mechanism of enterotoxigenic Escherichia coli and vaccines against it. Microb Pathog. 2018;117:162-169.
2. Alerasol M, Gargari SLM, Nazarian S, Bagheri S. 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.
3. Platts-Mills JA, Babji S, Bodhidatta L, Gratz J, Haque R, Havt A, et al. Pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED). Lancet Glob Health 2015;3: e564-75.
4. Tobias J, Svennerholm A-M, Carlin NI, Lebens M, Holmgren J. Construction of a non-toxigenic Escherichia coli oral vaccine strain expressing large amounts of CS6 and inducing strong intestinal and serum anti-CS6 antibody responses in mice. Vaccine 2011; 29:8863-8869.
5. Bagheri S, Gargari SLM, 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.
6. Nazarian S, Gargari SLM, 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 Methods 2012; 90:36-45.
7. Bourgeois AL, Wierzba TF, Walker RI. Status of vaccine research and development for enterotoxigenic Escherichia coli. Vaccine 2016; 34:2880-2886.
8. Fleckenstein JM, Hardwidge PR, Munson GP, Rasko DA, Sommerfelt H, Steinsland H. Molecular mechanisms of enterotoxigenic Escherichia coli infection. Microb Infect 2010; 12:89-98.
9. 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.
10. Walker RI, Wierzba TF, Mani S, Bourgeois AL. Vaccines against Shigella and enterotoxigenic Escherichia coli: A summary of the 2016 VASE Conference. Vaccine 2017;35:6775-6782.
11. Hajizade A, Ebrahimi F, Salmanian A-H, Arpanae A, Amani J. Nanoparticles in vaccine development. JABR 2015; 1:125-134.
12. Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Préat V. PLGA-based nanoparticles: an overview of biomedical applications. J Control Release 2012; 161:505-522.
13. Hans M, Lowman A. Biodegradable nanoparticles for drug delivery and targeting. Curr Opin Solid State Mater Sci 2002; 6:319-327.
14. Mendyk A, Jachowicz R, Fijorek K, Dorozynski P, Kulinowski P, Polak S. KinetDS: an open source software for dissolution test data analysis. Dissolut Technol 2012; 19:6-11.
15. Costa P, Lobo JMS. Modeling and comparison of dissolution profiles. Eur J Pharm Sci 2001; 13:123-133.
16. De SN, Chatterje D. An experimental study of the mechanism of action of Vibrio cholerae on the intestinal mucous membrane. J Pathol 1953; 66:559-562.
17. Matías J, Berzosa M, Pastor Y, Irache JM, Gamazo C. Maternal Vaccination. Immunization of Sows during Pregnancy against ETEC Infections. Vaccines (Basel) 2017;5:E48.
18. Ramazani F, Chen W, van Nostrum CF, Storm G, Kiessling F, Lammers T, et al. Strategies for encapsulation of small hydrophilic and amphiphilic drugs in PLGA microspheres: state-of-the-art and challenges. Int J Pharm 2016; 499:358-367.
19. Westedt U, Kalinowski M, Wittmar M, Merdan T, Unger F, Fuchs J, et al. Poly (vinyl alcohol)-graft-poly (lactide-co-glycolide) nanoparticles for local delivery of paclitaxel for restenosis treatment. J Control Release 2007; 119:41-51.
20. Kumari A, Yadav SK, Yadav SC. Biodegradable polymeric nanoparticles based drug delivery systems. Colloids Surf B Biointerfaces 2010; 75:1-18.
21. Sah E, Sah H. Recent trends in preparation of poly (lactide-co-glycolide) nanoparticles by mixing polymeric
organic solution with antisolvent. J Nanomater 2015;2015:1-22.
22. Papadimitriou S, Bikiaris D. Novel self-assembled core–shell nanoparticles based on crystalline amorphous moieties of aliphatic copolyesters for efficient controlled drug release. J Control Release 2009; 138:177-184.
23. Barratt G. Colloidal drug carriers: achievements and perspectives. Cell Mol Life Sci 2003; 60:21-37.
24. Rawat M, Singh D, Saraf S, Saraf S. Nanocarriers: promising vehicle for bioactive drugs. Biol Pharm Bull 2006; 29:1790-1798.
25. Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Préat V. PLGA-based nanoparticles: an overview of biomedical applications. J Control Release 2012; 161:505-522.
26. Pakulska MM, Donaghue IE, Obermeyer JM, Tuladhar A, McLaughlin CK, Shendruk TN, et al. Encapsulation-free controlled release: Electrostatic adsorption eliminates the need for protein encapsulation in PLGA nanoparticles. Sci Adv 2016; 2:e1600519.
27. Motulsky H, Christopoulos A. Fitting models to biological data using linear and nonlinear regression: a practical guide to curve fitting: Oxford University Press; 2004.
28. Singhvi G, Singh M. In vitro drug release characterization models. Int J Pharm Stud Res 2011; 2:77-84.
29. Pezzini BR, Grossl AD, Muraro A, Bazzo GC, Soares L. Formulation and in vitro assessment of sustained release matrix tablets of atenolol containing Kollidon SR and carnauba wax. Afr J Pharm Pharmacol 2014; 8:1058-1065.
30. Yan J, Wang Y, Shao SH, Mao YF, Li HW, Luo YH. Construction of prokaryotic expression system of ltB-ureB fusion gene and identification of the recombinant protein immunity and adjuvanticity. World J Gastroenterol 2004; 10:2675-2679.
31. Hajishengallis G, Arce S, Gockel C, Connell T, Russell M. Immunomodulation with enterotoxins for the generation of secretory immunity or tolerance: applications for oral infections. J Dent Res 2005; 84:1104-1116.
32. Salimian J, Salmanian A, Khalesi R, Mohseni M, Moazzeni S. Antibody against recombinant heat-labile enterotoxin B subunit (rLTB) could block LT binding to ganglioside M1 receptor. Iranian J Microbiol 2010; 2:120-127.
33. Gheibi Hayat SM, Mousavi Gargari SL, Nazarian S. Construction and immunogenic properties of a chimeric protein comprising CfaE, CfaB and LTB against Enterotoxigenic Escherichia coli. Biologicals. 2016; 44:503-510.
34. Noroozi N, Mousavi Gargari SL, Nazarian S, Sarvary S, Rezaei R. Immunogenicity of enterotoxigenic Escherichia coli outer membrane vesicles encapsulated in chitosan nanoparticles. Iran J Basic Med Sci 2018;21:284-291.
Iranian Journal of Basic Medical Sciences
Volume 21, Issue 5
May 2018
Pages 517-524

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