Immunogenic evaluation of chimeric recombinant protein against ETEC, EHEC and Shigella

Document Type: Original article

Authors

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

2 Department of Biology, Faculty of Sciences, Imam Hossein University, Tehran, Iran.

3 Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

Abstract

Diarrheal diseases still remain health problem worldwide and out of many bacteria responsible for, Shigella and pathogenic Escherichia cause the most diarrheas in the world. Shigellacause bacterial dysenteries and shigellosis through invasion where the most effective proteins for pathogenesis is Ipac. Critical virulence protein for ETEC infection is CFA/I with two subunits called cfab and cfae. . Attachment of EHEC is the main step of infection and the protein Intimin plays the key role in this function.  Protection against the vast majority of responsible pathogens of diarrheas requires development of the combination vaccine against Shigella, ETEC and EHEC. In the present study, a multisubunitprotein (CII) containing immunologically significant parts of CfaB, IpaC and Intimin was designed. The chimeric gene (CII) was codon optimized and analyzed with different bioinformatic servers, then synthesized and expressed in E. coli. Mice, Guinea pig and, Caco-2 Cell line were used as challenge models for EHEC, shigella and ETEC respectively. The chimeric protein induced significant immune response and therefore could be a suitable vaccine candidate against these three pathogens.

Keywords


1. Unicef. Diarrhoea: why children are still dying and what can be done. unicef org/media/files/Final_Diarrhoea_Report_October_2009_final pdf. 2010.

2.  Ghenghesh KS, Franka E, Tawil K, Abeid S, Ali M, Taher I, Tobgi R. Infectious acute diarrhea in Libyan children: causative agents, clinical features, treatment and prevention. Libyan J Infect Dis 2008;2:10Á9.

3. Barry EM, Wang J, Wu T, Davis T, Levine MM. Immunogenicity of multivalent Shigella-ETEC candidate vaccine strains in a guinea pig model. Vaccine 2006;24: 3727-3734.

4.  Venkatesan MM, Hartman AB, Newland JW, Ivanova VS, Hale TL, McDonough M, Butterton J. Construction, characterization, and animal testing of WRSd1, a Shigella dysenteriae 1 vaccine. Infect immunol 2002;70:2950-2958.

5.  Malaei F, Hesaraki M, Saadati M, Ahdi AM, Sadraeian M, Honari H, Nazarian S. Immunogenicity of a new recombinant IpaC from Shigella dysenteriae type I in Guinea Pig as a aaccine candidate. Iran J Immunol 2013;10:110-117.

6.  Suzuki T, Yoshikawa Y, Ashida H, Iwai H, Toyotome T, Matsui H, Sasakawa C. High vaccine efficacy against shigellosis of recombinant noninvasive Shigella mutant that expresses Yersinia invasin. J Immunol 2006;177:4709-4717.

7.  Schroeder GN, Hilbi H. Molecular pathogenesis of Shigella spp.: controlling host cell signaling, invasion, and death by type III secretion. Clin Microbiol Rev 2008; 21:134-156.

8.  Kueltzo LA, Osiecki J, Barker J, Picking WL, Ersoy B, Picking WD, Middaugh CR. Structure-function analysis of invasion plasmid antigen C (IpaC) from Shigella flexneri. J Biol Chem 2003;278:2792-2798.

9.  Terry CM, Picking WL, Birket SE, Flentie K, Hoffman BM, Barker JR, Picking WD. The C-terminus of IpaC is required for effector activities related to Shigella invasion of host cells. Microb Pathog 2008;45:282-289.

10. Hunter P. Drinking water and diarrhoeal disease due to Escherichia coli. J Water  Health 2003;1:65-72.

11. Yi Y, Ma Y, Gao F, Mao X, Peng H, Feng Y, Fan Z, Wang G, Guo G, Yan J. Crystal structure of EHEC intimin: insights into the complementarity between EPEC and EHEC. PLoS One 2010;5:e15285.

12. Amani J, Salmanian AH, Rafati S, Mousavi SL. Immunogenic properties of chimeric protein from espA, eae and tir genes of Escherichia coli O157: H7. Vaccine 2010;28:6923-6929.

13. Amani J, Mousavi SL, Rafati S, Salmanian AH. In silico analysis of chimeric espA, eae and tir fragments of Escherichia coli O157:H7 for oral immunogenic applications. Theor Biol Med Model 2009;6:28.

14. Dean-Nystrom EA, Gansheroff LJ, Mills M, Moon HW, O'Brien AD. Vaccination of pregnant dams with intiminO157 protects suckling piglets from Escherichia coli O157: H7 infection. Infect Immun 2002;70:2414-2418.

15. Nazarian S, Gargari SLM, 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.

16. 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 Methods 2012;90:36-45.

17. Bouzari S, Shahrokhi N, Dashti A, Janani A-R, Jafari A. Construction and evaluation of chimeric heat-labile toxin B subunit and N-terminal (1–75) fragment of colonization factor antigen I gene of enterotoxigenic Escherichia coli. Ann Microbiol 2010;60:255-2561.

18. Li Y-F, Poole S, Rasulova F, McVeigh AL, Savarino SJ, Xia D. Crystallization and preliminary X-ray diffraction analyses of several forms of the CfaB major subunit of enterotoxigenic Escherichia coli CFA/I fimbriae. Acta Crystallogr Sect F: Struct Biol Cryst Commun 2009;65:242-247.

19. 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;169:205-212.

20. Nagy G, Emo L, Pál T. Strategies for the development of vaccines conferring broad-spectrum protection. Int J Med Microbiol 2008;298:379-395.

21. Arai R, Ueda H, Kitayama A, Kamiya N, Nagamune T. Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng 2001; 14:529.

22. Khaloiee F, Pourfarzam P, Rasooli I, Amani J, Nazarian S, Mousavi SL. In silico analysis of chimeric recombinant immunogen against three diarrhea causing bacteria. JCMR 2013;5:65-74.

23. Wadolkowski EA, Burris JA, O'Brien AD. Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli O157: H7. Infect Immun 1990;58:2438-45.

24. Babiuk S, Asper DJ, Rogan D, Mutwiri GK, Potter AA. Subcutaneous and intranasal immunization with type III secreted proteins can prevent colonization and shedding of Escherichia coli O157: H7 in mice. Microbial pathog. 2008;45:7-11.

25. Potter AA, Klashinsky S, Li Y, Frey E, Townsend H, Rogan D, Erickson G, Hinkley S, Klopfenstein T, Moxley RA. Decreased shedding of Escherichia coli O157: H7 by cattle following vaccination with type III secreted proteins. Vaccine 2004;22:362-369.

26. Adamus G, Mulczyk M, Witkowska D, Romanowska E. Protection against keratoconjunctivitis shigellosa induced by immunization with outer membrane proteins of Shigella spp. Infect  Immun 1980;30:321-324.

27. Hartman AB, Van De Verg LL, Collins HH, Tang DB, Bendiuk NO, Taylor DN, Powell CJ. Local immune response and protection in the guinea pig keratoconjunctivitis model following immunization with Shigella vaccines. Infect Immun 1994;62:412-420.

28. Altboum Z, Barry EM, Losonsky G, Galen JE, Levine MM. Microbial immunity and vaccines-Attenuated Shigella flexneri 2a DguaBA Strain CVD 1204 Expressing Enterotoxigenic Escherichia coli (ETEC) CS2 and CS3 Fimbriae as a Live Mucosal Vaccine against. Infec Immun 2001;69:3150-3158.

29. Lee J-Y, Yu J, Henderson D, Langridge WH. Plant-synthesized E. coli CFA/I fimbrial protein protects Caco-2 cells from bacterial attachment. Vaccine 2004;23: 222-231.

30. Cook S, Maiti P, DeVinney R, Allen‐Vercoe E, Bach S, McAllister T. Avian‐and mammalian‐derived antibodies against adherence‐associated proteins inhibit host cell colonization by Escherichia coli O157: H7. J Appl Microbiol 2007;103:1206-1219.

31. Oliveira IRd, Bessler HC, Bao SN, Lima RdL, Giugliano LG. Inhibition of enterotoxigenic Escherichia coli (ETEC) adhesion to Caco-2 cells by human milk and its immunoglobulin and non-immunoglobulin fractions. Braz J Microbiol 2007; 38:86-92.

32. Ahs JW, Tao W, Löfgren J, Forsberg BC. Diarrheal diseases in low-and middle-income countries: incidence, prevention and management. Open Infect Dis J 2010;4:113-124.

33. Karpman D, Békássy ZD, Sjögren A-C, Dubois MS, Karmali MA, Mascarenhas M, Jarvis KG, Gansheroff LJ, O’Brien AD, Arbus GS. Antibodies to intimin and Escherichia coli secreted proteins A and B in patients with enterohemorrhagic Escherichia coli infections. Pediatr Nephrol 2002;17:201-11.

34. Donnenberg MS, Tzipori S, McKee M, O'brien A, Alroy J, Kaper J. The role of the eae gene of enterohemorrhagic Escherichia coli in intimate attachment in vitro and in a porcine model. J Clin Invest 1993;92:1418.

35. Son W-G, Graham T, Gannon V. Immunological characterization of Escherichia coli O157: H7 intimin γ1. Clin Diagn Lab Immunol 2002;9:46-53.

36. Kühne SA, Hawes WS, La Ragione RM, Woodward MJ, Whitelam GC, Gough KC. Isolation of recombinant antibodies against EspA and intimin of Escherichia coli O157: H7. J Clin Microbiol 2004;42:2966-2976.

36. Khare S, Alali W, Zhang S, Hunter D, Pugh R, Fang FC, Libby SJ, Adams LG. Vaccination with attenuated Salmonella enterica Dublin expressing E coli O157: H7 outer membrane protein Intimin induces transient reduction of fecal shedding of E coli O157: H7 in cattle. BMC Vet Res 2010;6:35.

37. Kühne SA, Hawes WS, La Ragione RM, Woodward MJ, Whitelam GC, Gough KC. Isolation of recombinant antibodies against EspA and intimin of Escherichia coli O157: H7. J Clin Microbiol 2004;42:2966-2976.

38. Carvalho HM, Teel LD, Kokai-Kun JF, O'Brien AD. Antibody against the carboxyl terminus of intimin α reduces enteropathogenic Escherichia coli adherence to tissue culture cells and subsequent induction of actin polymerization. Infect Immun 2005; 73:2541-2546.

39. Schunk MK, Macallum GE. Applications and optimization of immunization procedures. ILAR J 2005;46:241-257.

40. Cooper HM, Paterson Y. Production of polyclonal antisera. Curr Protoc Neurosci 2009;Chapter 5(Unit 5.5):10.

41. Yoshida M, Claypool SM, Wagner JS, Mizoguchi E, Mizoguchi A, Roopenian DC, Lencer WI, Blumberg RS. Human neonatal Fc receptor mediates transport of IgG into luminal secretions for delivery of antigens to mucosal dendritic cells. Immunity 2004;20:769-783.

42. Hartman AB, Powell C, Schultz C, Oaks E, Eckels K. Small-animal model to measure efficacy and immunogenicity of Shigella vaccine strains. Infect Immun 1991;59:4075-4083.

43. Wang X, Guan Q, Wang X, Teng D, Mao R, Yao J, Wang J. Paving the way to construct a new vaccine against Escherichia coli from its recombinant outer membrane protein C via a murine model. Process Biochem 2015;50:1194-1201.

44. Gohar A, Abdeltawab NF, Fahmy A, Amin MA. Development of safe, effective and immunogenic vaccine candidate for diarrheagenic Escherichia coli main pathotypes in a mouse model. BMC Res Notes 2016;9:1.