title: Effect of codon optimization and subcellular targeting on Toxoplasma gondii antigen SAG1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice.
abstract: BACKGROUND
  Codon optimization and subcellular targeting were studied with the aim to increase the expression levels of the SAG178-322 antigen of Toxoplasma gondii in tobacco leaves. The expression of the tobacco-optimized and native versions of the SAG1 gene was explored by transient expression from the Agrobacterium tumefaciens binary expression vector, which allows targeting the recombinant protein to the endoplasmic reticulum (ER) and the apoplast. Finally, mice were subcutaneously and orally immunized with leaf extracts-SAG1 and the strategy of prime boost with rSAG1 expressed in Escherichia coli was used to optimize the oral immunization with leaf extracts-SAG1.
  
  
  RESULTS
  Leaves agroinfiltrated with an unmodified SAG1 gene accumulated 5- to 10-fold more than leaves agroinfiltrated with a codon-optimized SAG1 gene. ER localization allowed the accumulation of higher levels of native SAG1. However, no significant differences were observed between the mRNA accumulations of the different versions of SAG1. Subcutaneous immunization with leaf extracts-SAG1 (SAG1) protected mice against an oral challenge with a non-lethal cyst dose, and this effect could be associated with the secretion of significant levels of IFN-gamma. The protection was increased when mice were ID boosted with rSAG1 (SAG1+boost). This group elicited a significant Th1 humoral and cellular immune response characterized by high levels of IFN-gamma. In an oral immunization assay, the SAG1+boost group showed a significantly lower brain cyst burden compared to the rest of the groups.
  
  
  CONCLUSIONS
  Transient agroinfiltration was useful for the expression of all of the recombinant proteins tested. Our results support the usefulness of endoplasmic reticulum signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The results showed that this plant-produced protein has potential for use as vaccine and provides a potential means for protecting humans and animals against toxoplasmosis.
authors.affiliation: IIB-INTECH, Camino de Circunvalación km 6, Provincia de Buenos Aires, Argentina.
authors.forename: Melina
authors.initials: M
authors.lastname: Laguía-Becher
authors.forename: Valentina
authors.initials: V
authors.lastname: Martín
authors.forename: Mauricio
authors.initials: M
authors.lastname: Kraemer
authors.forename: Mariana
authors.initials: M
authors.lastname: Corigliano
authors.forename: María L
authors.initials: ML
authors.lastname: Yacono
authors.forename: Alejandra
authors.initials: A
authors.lastname: Goldman
authors.forename: Marina
authors.initials: M
authors.lastname: Clemente
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan / Q000276:immunology*; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D005260:Female; D007111:Immunity, Cellular; D056724:Immunity, Humoral; D007114:Immunization / Q000379:methods; D007371:Interferon-gamma / Q000276:immunology; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000276:immunology; D015800:Protozoan Proteins / Q000276:immunology*; D016052:Protozoan Vaccines / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D018417:Th1 Cells / Q000276:immunology; D014026:Nicotiana / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014122:Toxoplasma / Q000276:immunology; D014124:Toxoplasmosis, Animal / Q000276:immunology / Q000517:prevention & control*
chemical_list: D000953:Antigens, Protozoan; D003062:Codon; D015800:Protozoan Proteins; D016052:Protozoan Vaccines; D011994:Recombinant Proteins; C047168:SAG1 antigen, Toxoplasma; D007371:Interferon-gamma