Molecular Biotechnology

, Volume 32, Issue 1, pp 31–42 | Cite as

Analysis of immune response in young and aged mice vaccinated with corn-derived antigen against Escherichia coli heat-labile enterotoxin

  • Sule Karaman
  • Joan Cunnick
  • Kan Wang


Enterotoxigenic strains of Escherichia coli produce a heat-labile holotoxin (LT), which causes diarrhea. We engineered corn seeds to produce LT-B, the nontoxic subunit of LT, to serve as a plant-derived vaccine to traveler's diarrhea and as an adjuvant for co-administered proteins. We previously demonstrated that a strong mucosal and systemic antibody response is elicited in young mice with oral administration of corn-derived LT-B. The present study examined systemic and mucosal antibody responses to LT-B in young and aged mice, and recall responses to oral administration and injection of LT-B in aged mice. Specific IgA and IgG antibodies were detectable during an 11-mo period, although the concentration of antigen-specific antibodies declined gradually. Booster by feeding or injection dramatically increased the concentration of specific IgA from that seen in young mice. Specific IgG levels were boosted to concentrations similar to those in young mice. This effect may be age-dependent and related to prior immunization exposure. Analysis of the antibody response of naive aged mice against corn-derived LT-B demonstrated an age-related suppression in specific IgG production, but not specific IgA. These results may provide important information for edible vaccine strategies for young and aged individuals.

Index Entries

Plant-derived vaccines immune memory LT-B 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    McGhee, J. R. and Kyono, H. (1998) Mucosal immunology. In Fundamental Immunology (Paul, W. E., ed.) San Diego, Academic Press, pp. 909–945.Google Scholar
  2. 2.
    Wang, K., Chikwamba, R., and Cunnick, J. (2004) Plant-based oral vaccines. In Novel Vaccination Strategies (Kaufmann, S. H. E., ed.) Weinheim, Germany, Wiley-VCH, pp. 387–411.CrossRefGoogle Scholar
  3. 3.
    Tacket, C. O., Mason, H. S., Losonsky G., Clements, J. D., Levine, M. M., and Arntzen, C. J. (1998) Immunogenicity in humans of a recombinat bacterial-antigen delivered in transgenic potato. Nature Med. 4, 607–609.PubMedCrossRefGoogle Scholar
  4. 4.
    Tacket, C. O., Mason, H. S., Losonsky, G., Estes, M. K., Levine, M. M., and Arntzen, C. J. (2000) Human immune responses to a novel Norwalk virus vaccine delivered in transgenic potatoes. J. Infect. Dis. 182, 302–305.PubMedCrossRefGoogle Scholar
  5. 5.
    Kapusta, J., Modelska, A., Figlerowicz, M., et al. (1999) A plant-derived edible vaccine against hepatitis B virus. FASEB J. 13, 1796–1799.PubMedGoogle Scholar
  6. 6.
    Fujihashi, K., Koga, T., and McGhee, J. R. (2000) Mucosal vaccination and immune responses in the elderly. Vaccine 18, 1675–1680.PubMedCrossRefGoogle Scholar
  7. 7.
    Kanellos, T. S., Byarugaba, D. K., Russell, P. H., Howard, C. R., and Partidos, C. D. (2000) Naked DNA when co-administered intranaslly with heatlabile enterotoxin of Escherichia coli primes effectively for systemic B- and T-cell responses to the encoded antigen. Immunol. Lett. 74, 215–220.PubMedCrossRefGoogle Scholar
  8. 8.
    Choi, A. H., McNeal, M. M., Flint, J. A., et al. (2000) The level of protection against rotavirus shedding in mice following immunization with a chimeric VP6 protein is dependent on the route and the co-administered adjuvant. Vaccine 20, 1733–1740.CrossRefGoogle Scholar
  9. 9.
    Elson, C. O., and Ealding W. (1984) Generalized systemic mucosal immunity in mice after mucosal stimulation with cholera toxin. J. Immunol. 132, 2736–2741.PubMedGoogle Scholar
  10. 10.
    Clements, J. D., Hartzog, N. M., and Lyon, F. L. (1998) Adjuvant activity of E. coli heat labile enterotoxin and effect on the induction of oral tolerance in, mice to unrelated protein antigens. Vaccine 6, 269–277.CrossRefGoogle Scholar
  11. 11.
    Xu-Amano, J., Kiyono, H., Jackson, R. J., et al. (1993) Helper T cell subset for immunoglobulin A responses: oral immunization with tetanus toxoid and cholera toxin as adjuvant selectively induces Th2 cells in mucosa associated tissues. J. Exp. Med. 178, 1309–1320.PubMedCrossRefGoogle Scholar
  12. 12.
    Haan, L., Holtrop, M., Verweij W. R., Agsteribbe, E., and Wilschut, J. (1996) Mucosal immunogenity of the Escherichia coli heat-lable enterotoxin: role of the A subunit. Vaccine 14, 260–266.PubMedCrossRefGoogle Scholar
  13. 13.
    Millar, D. G., Hirst, T. R., and Snider, D. P. (2001) Escherichia coli heat-labile enterotxin B subunit is a more potent mucosal adjuvant than its closely related homologue, the B subunit of cholera toxin. Infect. Immunity 69, 3476–3482.CrossRefGoogle Scholar
  14. 14.
    Pizza, M., Domenighin, M., Hol, W., t al. (1994) Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutanesis. Mol. Microbiol. 14, 51–60.PubMedCrossRefGoogle Scholar
  15. 15.
    Verweij, W. R., Haan, L., Holtrop, M., et al. (1998) Mucosal immunoadjuvant activity of Escherichia coli heat-labile enterotoxin and its B subunit: induction of systemic IgG and secretory IgA responses in mice by intranasal immunization with influenza virus surface antigen. Vaccine 16, 2069–2076.PubMedCrossRefGoogle Scholar
  16. 16.
    Mason, H. S., Haq, T. A., Clements, J. D., and Arntzen, C. J. (1998) Edible vaccine protects mice against Escherichia coli heat labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene. Vaccine 16, 1336–1343.PubMedCrossRefGoogle Scholar
  17. 17.
    Chikwamba R., Cunnick, J., Hathaway, D., McMurray, J., Mason, H., and 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.PubMedCrossRefGoogle Scholar
  18. 18.
    Guidry, J. J., Cardenas, L., Cheng, E., and Clements, J. D. (1997) Role of receptor binding in toxicity, immunogenicity, and adjuvanticity of Escherichia coli heat-labile enterotoxin. Infect. Immunol. 65, 4943–4950.Google Scholar
  19. 19.
    Mayer, L. (2000) Oral tolerance: new approaches, new problems. Clin. Immunol. 94, 1–8.PubMedCrossRefGoogle Scholar
  20. 20.
    Czerkinsky, C., Anjuere, F., McGhee, J. R., et al. (1999) Mucosal immunity and tolerance: relevance to vaccine development. Immunol. Rev. 170, 197–222.PubMedCrossRefGoogle Scholar
  21. 21.
    Nagler-Anderson, C., Bober, L. A., Robinson, M. E., Sikind, G. W., and Thorbecke, G. J. (1986) Suppression of type YY collagen-induced arthritis by intragastric administration of soluble type II collagen. Proc. Natl. Acad. Sci. USA 83, 7443–7446.PubMedCrossRefGoogle Scholar
  22. 22.
    Nedrud, J. G. and Sigmund, N. (1991) Cholera toxin as a mucosal adjuvant: II. Antibody responses to non-target dietary antigens are not increased. Regional Immunol. 3, 217–222.Google Scholar
  23. 23.
    Brennan, F. R., Bellaby, T., Helliwell, S. M., et al. (1999) Chimeric plant virus particles administered nasally or orally induce systemic and mucosal immune responses in mice. J. Virol. 73, 930–938.PubMedGoogle Scholar
  24. 24.
    Tuboly, T., Yu, W., Bailey, A., et al. (2000) Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants. Vaccine 18, 2023–2038.PubMedCrossRefGoogle Scholar
  25. 25.
    Yu, J. and Langridge, H. R. (2001) A plant-based multicomponent vaccine protects mice from enteric diseases. Nature Biotech. 19, 548–552.CrossRefGoogle Scholar
  26. 26.
    Lauterslager, T. G. M., Florack, D. E. A., van der Wal, T. J., et al. (2001) Oral immunization of naïve and primed animals with transgenic potato tuber expressing LT-B. Vaccine 19, 2749–2755.PubMedCrossRefGoogle Scholar
  27. 27.
    Yu, J. and Langridge, H. R. (2003) Expression of rotavirus capsid protein VP6 in transgenic potato and its oral immunogenicity in mice. Transgenic Res. 12, 163–169.PubMedCrossRefGoogle Scholar
  28. 28.
    Ma, Y., Chen, Q., and Ross, A. C. (2005) Retinoic acid and polyriboinosinic: polyribocytidylic acid stimulate robust anti-tetanus antibody production while differentially regulating type 1/type 2 cytokines and lymphocyte populations. J. Immunol. 174, 7961–7969.PubMedGoogle Scholar
  29. 29.
    Clements, J. D. (1990) Construction of a nontoxic fusion peptide for immunization against Escherichia coli strains that produce heat-labile and heat-stable enterotoxins. Infect. Immunol. 58, 1159–1166.Google Scholar
  30. 30.
    Wakikawa, A., Utsuyama, M., and Hirokawa, K. (1997) Altered expression of various receptors on T cells in young and old mice after mitogenic stimulation: a flow cytometric analysis. Mech. Ageing Dev. 94, 113–122.PubMedCrossRefGoogle Scholar
  31. 31.
    Rabinowich, H., Goses, Y., Reshef, T., and Klajman, A. (1985) Interleukin-2 production and activity in aged humans. Mech. Ageing Dev. 32, 213–226.PubMedCrossRefGoogle Scholar
  32. 32.
    Kurashima, C., Utsuyama, M, Kasai, M, Ishijama, S. A., Konno, A., and Hirokawa, K. (1995) The role of thymus in the aging of Th cell subpopulations and age-associated alteration of cytokine production by these cells. Int. Immunol. 7, 97–104.PubMedCrossRefGoogle Scholar
  33. 33.
    Kaplan, C., Valdez, J. C., Chandrasekaran, R., et al (2002) Th1 and Th2 cytokines regulate proteoglycan-specific autoantibody isotypes and arthiritis. Arthritis Res. 4, 54–58.PubMedCrossRefGoogle Scholar
  34. 34.
    Hiroi, T., Fujihashi, K., McGhee, J. R., and Kiyono, H. (1995) Polarized Th2 cytokine expression by both mucosal gamma delta and alpha beta T cells. Eur. J. Immunol. 25, 2743–2751.PubMedCrossRefGoogle Scholar
  35. 35.
    Schmuker, D. and Owen, R. (1997) Aging and gastrointestinal mucosal immune response. Mech. Ageing Dev. 13, 534–541.Google Scholar
  36. 36.
    Koga, T., McGhee, J. R., Kato, H., Kato, R., Kiyono, H., and Fujihashi, K. (2000) Evidence for early aging in the mucosal immune system. J. Immunol. 165, 5352–5359.PubMedGoogle Scholar
  37. 37.
    Arranz, E. and Ferguson, A. (1992) Aging and gastrointestina immunity. Gastroenterol. Hepatol. 4, 1–14.Google Scholar
  38. 38.
    Beherkaka, A. A., Paiva, S., Leka, L. S., Ribaya-Mercado, J. D., Russell, R. M., and Meydani, S. N. (2001) Effect of age on the gastrointestinal-associated mucosal immune response of humans. J. Geronol. 56A, B218-B223.Google Scholar

Copyright information

© Humana Press Inc 2006

Authors and Affiliations

  1. 1.Plant Transformation Facility, Department of AgronomyIowa State UniversityAmes
  2. 2.Interdepartment Plant Physiology and MajorIowa State UniversityAmes
  3. 3.Department of Animal ScienceIowa State UniversityAmes

Personalised recommendations