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

MELINA LAGUIA-BECHER; VALENTINA MARTIN; MAURICIO KRAEMER; MARIANA CORIGLIANO; MARIA L. YACONO; ALEJANDRA GOLDMAN; MARINA CLEMENTE

BMC BIOTECHNOLOGY

BIOMED CENTRAL LTD

Año: 2010 vol. 10 p. 52 - 65

1472-6750

Background: Codon optimization and subcellular targeting were studied with the aim toincrease the expression levels of the SAG178-322 antigen of Toxoplasma gondii in tobaccoleaves. The expression of the tobacco-optimized and native versions of the SAG1 gene wasexplored by transient expression from the Agrobacterium tumefaciens binary expressionvector, which allows targeting the recombinant protein to the endoplasmic reticulum (ER)and the apoplast. Finally, mice were subcutaneously and orally immunized with leafextracts-SAG1 and the strategy of prime boost with rSAG1 expressed in Escherichia coliwas used to optimize the oral immunization with leaf extracts-SAG1.Results: Leaves agroinfiltrated with an unmodified SAG1 gene accumulated 5- to 10-foldmore than leaves agroinfiltrated with a codon-optimized SAG1 gene. ER localizationallowed the accumulation of higher levels of native SAG1. However, no significantdifferences were observed between the mRNA accumulations of the different versions ofSAG1. Subcutaneous immunization with leaf extracts-SAG1 (SAG1) protected miceagainst an oral challenge with a non-lethal cyst dose, and this effect could be associatedwith the secretion of significant levels of IFN-. The protection was increased when micewere ID boosted with rSAG1 (SAG1+boost). This group elicited a significant Th1 humoraland cellular immune response characterized by high levels of IFN-. In an oralimmunization assay, the SAG1+boost group showed a significantly lower brain cyst burdencompared to the rest of the groups.Conclusion: Transient agroinfiltration was useful for the expression of all of therecombinant proteins tested. Our results support the usefulness of endoplasmic reticulumsignal peptides in enhancing the production of recombinant proteins meant for use asvaccines. The results showed that this plant-produced protein has potential for use asvaccine and provides a potential means for protecting humans and animals againsttoxoplasmosis.