Production of hybrid-IgG/IgA plantibodies with neutralizing activity against Shiga toxin 1

PLoS One. 2013 Nov 28;8(11):e80712. doi: 10.1371/journal.pone.0080712. eCollection 2013.

Abstract

Shiga toxin 1 (Stx1) is a virulence factor of enterohemorrhagic Escherichia coli, such as the O157:H7 strain. In the intestines, secretory IgA (SIgA) is a major component of the immune defense against pathogens and toxins. To form SIgA, the production of dimeric IgA that retains biological activity is an important step. We previously established hybrid-IgG/IgA having variable regions of the IgG specific for the binding subunit of Stx1 (Stx1B) and the heavy chain constant region of IgA. If hybrid-IgG/IgA cDNAs can be expressed in plants, therapeutic or preventive effects may be expected in people eating those plants containing a "plantibody". Here, we established transgenic Arabidopsis thaliana expressing dimeric hybrid-IgG/IgA. The heavy and light chain genes were placed under the control of a bidirectional promoter and terminator of the chlorophyll a/b-binding protein of Arabidopsis thaliana (expression cassette). This expression cassette and the J chain gene were subcloned into a single binary vector, which was then introduced into A. thaliana by means of the Agrobacterium method. Expression and assembly of the dimeric hybrid-IgG/IgA in plants were revealed by ELISA and immunoblotting. The hybrid-IgG/IgA bound to Stx1B and inhibited Stx1B binding to Gb3, as demonstrated by ELISA. When Stx1 holotoxin was pre-treated with the resulting plantibody, the cytotoxicity of Stx1 was inhibited. The toxin neutralization was also demonstrated by means of several assays including Stx1-induced phosphatidylserine translocation on the plasma membrane, caspase-3 activation and 180 base-pair DNA ladder formation due to inter-nucleosomal cleavage. These results indicate that edible plants containing hybrid-IgG/IgA against Stx1B have the potential to be used for immunotherapy against Stx1-caused food poisoning.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Blocking / genetics
  • Antibodies, Blocking / immunology
  • Antigens, Tumor-Associated, Carbohydrate / immunology
  • Antigens, Tumor-Associated, Carbohydrate / metabolism
  • Arabidopsis / genetics
  • Arabidopsis / metabolism
  • Cell Death / immunology
  • Gene Expression
  • Gene Order
  • Immunoglobulin A / genetics
  • Immunoglobulin A / immunology*
  • Immunoglobulin G / genetics
  • Immunoglobulin G / immunology*
  • Plantibodies / genetics
  • Plantibodies / immunology*
  • Plants, Genetically Modified
  • Protein Binding / immunology
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Shiga Toxin 1 / immunology*
  • Shiga Toxin 1 / metabolism

Substances

  • Antibodies, Blocking
  • Antigens, Tumor-Associated, Carbohydrate
  • Gb3 antigen
  • Immunoglobulin A
  • Immunoglobulin G
  • Plantibodies
  • Recombinant Proteins
  • Shiga Toxin 1

Grants and funding

This work was partly supported by a grant-in-aid (16590055, 23659067, 25670063) and by research funding for the Global COE Program from the Japan Society for the Promotion of Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.