Skip to main content

Advertisement

SpringerLink
Log in

Expression of the cholera toxin B subunit (CT-B) in maize seeds and a combined mucosal treatment against cholera and traveler’s diarrhea

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

The non-toxic B subunit (CT-B) of cholera toxin from Vibrio cholerae is a strong immunogen and amplifies the immune reaction to conjugated antigens. In this work, a synthetic gene encoding for CT-B was expressed under control of a γ-zein promoter in maize seeds. Levels of CT-B in maize plants were determined via ganglioside dependent ELISA. The highest expression level recorded in T1 generation seeds was 0.0014% of total aqueous soluble protein (TASP). Expression level of the same event in the T2 generation was significantly increased to 0.0197% of TASP. Immunogenicity of maize derived CT-B was evaluated in mice with an oral immunization trial. Anti-CTB IgG and anti-CTB IgA were detected in the sera and fecal samples of the orally immunized mice, respectively. The mice were protected against holotoxin challenge with CT. An additional group of mice was administrated with an equal amount (5 μg per dose each) of mixed maize-derived CT-B and LT-B (B subunit of E. coli heat labile toxin). In the sera and fecal samples obtained from this group, the specific antibody levels were enhanced compared to either the same or a higher amount of CT-B alone. These results suggest that a synergistic action may be achieved using a CT-B and LT-B mixture that can lead to a more efficacious combined vaccine to target diarrhea induced by both cholera and enterotoxigenic strains of Escherichia coli.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Arakawa T, Chong DKX, Merrit JL, Langridge WHR (1997) Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Res 6:403–413

    Article  PubMed  CAS  Google Scholar 

  • Arakawa T, Chong DKX, Langridge WH (1998) Efficacy of a food plant-based oral cholera toxin B subunit vaccine. Nat Biotech 16:292–297

    Article  CAS  Google Scholar 

  • Blake MS, Johnston KH, Russell-Jones GJ, Gotschlich EC (1984) A rapid sensitive method for detection of alkaline phosphatase conjugated antibody on Western blots. Anal Biochem 136:175–179

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Brown LR, Halwell B (1998) China’s water shortage could shake world food security. World Watch 11:10–16

    PubMed  CAS  Google Scholar 

  • Chikwamba RK, Cunnick J, Hathaway D, McMurray J, Mason H, Wang K (2002) A functional antigen in a practical crop: LT-B producing maize protects mice against Escherichia coli heat labile enterotoxin (LT) and Cholera toxin (CT). Transgenic Res 11:479–493

    Article  PubMed  CAS  Google Scholar 

  • Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5:213–218

    Article  PubMed  CAS  Google Scholar 

  • Dertzbough MT, Elson CO (1993) Comparative effectiveness of the cholera toxin B subunit and alkaline phosphatase as carriers for oral vaccines. Infect Immun 61:48–55

    Google Scholar 

  • Frame BR, Zhang H, Cocciolone SM, Sidorenko LV, Dietrich CR, Pegg SE, Zhen S, Schnable PS, Wang K (2000) Production of transgenic maize from bombarded type II callus: effect of gold particle size and callus morphology on transformation efficiency. In Vitro Cell Dev Biol-Plant 36:21–29

    Article  Google Scholar 

  • Gallie DR, Tanguay RL, Leathers V (1995) The tobacco etch viral 5′ leader and poly(A) tail are functionally synergistic regulators of translation. Gene 165:233–238

    Article  PubMed  CAS  Google Scholar 

  • George-Chandy A, Eriksson K, Lebens M, Nordström I, Schön E, Holmgren J (2001) Cholera toxin B subunit as a carrier molecule promotes antigen presentation and increases CD40 and CD86 expression on antigen-presenting cells. Infect Immun 69:5716–5725

    Article  PubMed  CAS  Google Scholar 

  • Girard MP, Steele D, Chaignat CL, Kieny MP (2006) A review of vaccine research and development: human enteric infections. Vaccine 24:2732–2750

    Article  PubMed  CAS  Google Scholar 

  • Guidry JJ, Cardenas L, Cheng E, Clements JD (1997) Role of receptor binding in toxicity, immunogenicity and adjuvanticity of Escherichia coli heat labile enterotoxin. Infect Immun 65:4943–4950

    PubMed  CAS  Google Scholar 

  • Holmgren J, Czerkinsky C (2005) Mucosal immunity and vaccines. Nat Med 11:S45–S53

    Article  PubMed  CAS  Google Scholar 

  • Hood EE, Witcher DR, Maddock S, Meyer T, Baszczynski C, Bailey M, Flynn P, Register J, Marshall L, Bond D, Kulisek E, Kusnadi A, Evangelista R, Nikolov Z, Wooge C, Mehigh RJ, Hernan R, Kappel WK, Ritland D, Li CP, Howard JA (1997) Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification. Mol Breed 3:291–306

    Article  CAS  Google Scholar 

  • Jani D, Meena LS, Rizwan-ul-Haq QM, Singh Y, Sharma AK, Tyagi AK (2002) Expression of cholera toxin B subunit in transgenic tomato plants. Transgenic Res 11:447–454

    Article  PubMed  CAS  Google Scholar 

  • Jani D, Singh NK, Chattacharya S, Meena LS, Singh Y, Upadhyay SN, Sharma AK, Tyagi AK (2004) Studies on the immunogenic potential of plant-expressed cholera toxin B subunit. Plant Cell Rep 22:471–477

    Article  PubMed  CAS  Google Scholar 

  • Karaman S, Cunnick J, Wang K (2006) Analysis of immune response in young and aged mice vaccinated with corn-derived antigen against Eschericia coli heat-labile enterotoxin. Mol Biotech 32:31–42

    Article  CAS  Google Scholar 

  • Kim YS, Kim MY, Kim TG, Yang MS (2009) Expression and assembly of cholera toxin B subunit (CTB) in transgenic carrot (Daucuscarota L.). Mol Biotechnol 41:8–14

    Article  PubMed  Google Scholar 

  • Kuo TT, Baker K, Yoshida M, Qiao S, Aveson VG, Lencer WI, Blumberg RS (2010) Neonatal Fc receptor: from immunity to therapeutics. J Clin Immunol 30:777–789

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lebens M, Shahabi V, Backstrom M, Houze T, Lindblad N, Holmgren J (1996) Synthesis of hybrid molecules between heat-labile enterotoxin and cholera toxin B subunits: potential for use in a broad-spectrum vaccine. Infect Immun 64:2144–2150

    PubMed  CAS  Google Scholar 

  • Luci C, Hervouet C, Rousseau D, Holmgren J, Czerkinsky C, Anjuère F (2006) Dendritic cell-mediated induction of mucosal cytotoxic responses following intravaginal immunization with the nontoxic B subunit of cholera toxin. J Immunol 176:2749–2757

    PubMed  CAS  Google Scholar 

  • Ma JK, Drake PM, Christou P (2003) The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4:794–805

    Article  PubMed  CAS  Google Scholar 

  • Marks MD, Lindell JS, Larkins BA (1985) Quantitative analysis of the accumulation of zein mRNA during endosperm development. J Biol Chem 260:16445–16450

    PubMed  CAS  Google Scholar 

  • Mason HS, Haq TA, Clements JD, Arntzen CJ (1998) Edible vaccine protects mice against Escherichia coli heat labile enterotoxin (LT): Potatoes expressing a synthetic LT-B gene. Vaccine 16:1336–1343

    Article  PubMed  CAS  Google Scholar 

  • Mason HS, Warzecha H, Mor TS, Arntzen CJ (2002) Edible plant vaccines: applications for prophylactic and therapeutic molecular medicine. Trends Mol Med 8:324–329

    Article  PubMed  CAS  Google Scholar 

  • Moeller L, Gan Q, Wang K (2009) A bacterial signal peptide is functional in plants and directs proteins to the secretory pathway. J Exp Bot 60:3337–3352

    Article  PubMed  CAS  Google Scholar 

  • Nochi T, Takagi H, Yuki Y, Yang L, Masumura T, Mejima M, Nakanishi U, Matsumura A, Uozumi A, Hiroi T, Morita S, Tanaka K, Takaiwa F, Kiyono H (2007) Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination. Proc Natl Acad Sci USA 104:10986–10991

    Article  PubMed  CAS  Google Scholar 

  • Qadri F, Ahmed T, Ahmed F, Bradley Sack R, Sack DA, Svennerholm AM (2003) Safety and immunogenicity of an oral, inactivated enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine in Bangladeshi children 18–36 months of age. Vaccine 2:2394–2403

    Article  Google Scholar 

  • Rhee Y, Staswick PE (1992) Nucleotide sequence for the soybean vegetative storage protein vspB gene. Plant Physiol 98:794–795

    Article  PubMed  CAS  Google Scholar 

  • Richardson SH, Giles JC, Kriger KS (1984) Sealed adult mice: a new model for enterotoxin evaluation. Infect Immun 43:482–486

    PubMed  CAS  Google Scholar 

  • Rigano MM, Sala F, Arntzen CJ, Walmsley AM (2003) Targeting of plant-derived vaccine antigens to immunoresponsive mucosal sites. Vaccine 21:809–811

    Article  PubMed  CAS  Google Scholar 

  • Roopenian DC, Akilesh S (2007) FcRn: the neonatal Fc receptor comes of age. Nat Immunol 7:715–725

    Article  CAS  Google Scholar 

  • Rosales-Mendoza S, Sora-Guerra RE, López-Revilla R, Moreno-Fierros L, Alpuche-Solís ÁG (2008) Ingestion of transgenic carrots expressing the Eschericia coli heat-labile enterotoxin B subunit protects mice against cholera toxin challenge. Plant Cell Rep 27:79–84

    Article  PubMed  CAS  Google Scholar 

  • Ruddock LW, Ruston SP, Kelly SM, Price NC, Freedman RB, Hirst TR (1995) Kinetics of acid-mediated disassembly of the B subunit pentamer of Escherichia coli heat-labile enterotoxin. J Biol Chem 270:29953–29958

    Article  PubMed  CAS  Google Scholar 

  • Sadeghi H, Bregenholt S, Wegmann D, Peterson JS, Holmgren J, Lebens M (2002) Genetic fusion of human insulin B-chain to the B subunit of cholera toxin enhances in vitro antigen presentation and induction of bystander suppression in vivo. Immunology 106:237–245

    Article  PubMed  CAS  Google Scholar 

  • Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA 81:8014–8018

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Frittsch EF, Maniatis TH (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, NY

    Google Scholar 

  • Sangtong V, Mottl EC, Long MJ, Lee M, Scott MP (2001) Serial extraction of endosperm drillings (SEED)—a method for detecting transgenes and proteins in single viable maize kernels (Protocols). Plant Mol Biol Rep 19:151–158

    Article  CAS  Google Scholar 

  • Savarino SJ, Hall ER, Bassily S, Wierzba TF, Youssef FG, Peruski LF Jr, Abu-Elyazeed R, Rao M, Francis WM, El Mohamady H, Safwat M, Naficy AB, Svennerholm AM, Jertborn M, Lee YJ, Clemens JD (2002) Pride Study Group. Introductory evaluation of an oral, killed whole cell enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine in Egyptian infants. Pediatr Infect Dis J 21:322–330

    Article  PubMed  Google Scholar 

  • Schaetti C (2009) Vaccines for enteric diseases: update on recent developments. Vaccine 8:1653–1655

    Google Scholar 

  • Shou S, Frame BR, Whitham S, Wang K (2004) Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation. Mol Breed 13:201–208

    Article  CAS  Google Scholar 

  • Tacket CO, Mason HS, Losonsky G, Clements JD, Levine MM, Arntzen CJ (1998) Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nat Med 4:607–609

    Article  PubMed  CAS  Google Scholar 

  • Tacket CO, Pasetti MF, Edelman R, Howard JA, Streatfield S (2004) Immunogenicity of recombinant LT-B delivered orally to humans in transgenic corn. Vaccine 22:4385–4389

    Article  PubMed  CAS  Google Scholar 

  • Tarkowski A, Sun JB, Holmdahl R, Holmgren J, Czerkinsky C (1999) Treatment of experimental autoimmune arthritis by nasal administration of a type II collagen-cholera toxoid conjugate vaccine. Arthritis Rheum 42:1628–1634

    Article  PubMed  CAS  Google Scholar 

  • Thompson CK, Movva NR, Tizard R, Crameri R, Davies JE, Lauwereys M, Botterman J (1987) Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. EMBO J 6:2519–2523

    PubMed  CAS  Google Scholar 

  • Tokuhara D, Yuki Y, Nochi T, Kodama T, Mejima M, Kurokawa S, Takahashi Y, Nanno M, Nakanishi U, Takaiwa F, Honda T, Kiyono H (2010) Secretory IgA-mediated protection against V. cholerae and heat-labile enterotoxin-producing enterotoxigenic Escherichia coli by rice-based vaccine. Proc Natl Acad Sci USA 107:8794–8799

    Article  PubMed  CAS  Google Scholar 

  • Williams NA, Hirst TR, Nashar TO (1999) Immune modulation by the cholera-like enterotoxins: from adjuvant to therapeutic. Immunol Today 20:95–101

    Article  PubMed  CAS  Google Scholar 

  • Zhang RG, Scott DL, Westbrook ML, Nance S, Spangler BD, Shipley GG, Westbrook EM (1995) The 2.4 A crystal structure of cholera toxin B subunit pentamer: choleragenoid. J Mol Biol 25:550–562

    Article  Google Scholar 

Download references

Acknowledgments

The authors wish to thank Iowa State University Plant Transformation Facility for providing maize transformation service. We are thankful to John Clements of Tulane University for providing bacterial LT-B and to Chad Niederhuth for his technical assistance. This work was partially supported by the United States Department of Agriculture (USDA-NRI 99–35504-7799) and Iowa State University Research Foundation to KW, the Biopharmaceutical/Bioindustrial Initiative of the Plant Sciences Institute at Iowa State University to KW and JC, and The Scientific and Technical Research Council of Turkey to SK.

Author information

Author notes

  1. S. Karaman

    Present address: Pioneer Hi-Bred International Inc., Ankeny, IA, 50021, USA

Authors and Affiliations

  1. Interdepartmental Plant Biology Major, Iowa State University, Ames, IA, 50011, USA

    S. Karaman

  2. Department of Animal Science, Iowa State University, Ames, IA, 50011, USA

    J. Cunnick

  3. Department of Agronomy, Iowa State University, G405 Agronomy Hall, Ames, IA, 50011-1010, USA

    K. Wang

Authors
  1. S. Karaman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. Cunnick
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K. Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to K. Wang.

Additional information

Communicated by P. Lakshmanan.

A contribution to the Special Issue: Plant Molecular Pharming in 2012 and Beyond.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Karaman, S., Cunnick, J. & Wang, K. Expression of the cholera toxin B subunit (CT-B) in maize seeds and a combined mucosal treatment against cholera and traveler’s diarrhea. Plant Cell Rep 31, 527–537 (2012). https://doi.org/10.1007/s00299-011-1146-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-011-1146-3

Keywords

Search

Navigation