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 Laguía-Becher,Mariana Corigliano,María L Yacono,Alejandra Goldman,Marina Clemente,Valentina Martín,Mauricio Kraemer

doi:10.1186/1472-6750-10-52

Melina Laguía-Becher, Mariana Corigliano + Show 5 more

Open Access

https://doi.org/10.1186/1472-6750-10-52

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Abstract

BackgroundCodon 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.ResultsLeaves 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-γ. 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-γ. In an oral immunization assay, the SAG1+boost group showed a significantly lower brain cyst burden compared to the rest of the groups.ConclusionTransient 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.

Highlights

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
A total of 57 codons were altered in the native SAG177-322 (nS) sequence to match the codon usage preferences of Nicotiana tabaccum, yielding an optimized-plant version of the SAG1 sequence named oS (Figure 1 and Table 1)
The final G + C content of the oS gene was lowered to 46% and the codon adaptation index value (CAI) was increased to 0.83 for matching with tobacco genes as compared to the 0.67 CAI value of the nS sequence

Summary

Introduction

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. Toxoplasma gondii is an obligate intracellular parasite capable of infecting a variety of mammals, including humans, and birds [5]. Toxoplasmosis is usually asymptomatic in healthy individuals. In pregnant women, congenital infection can cause severe neonatal malformations and ocular complications in the fetus; and in immunocompromised individuals, such as AIDS patients and transplant recipients, often results in fatal encephalitis [6,7]. Toxoplasmosis is of veterinary importance, especially in sheep and pigs, where it often results in abortion, causing considerable economic losses [8,9]. The tissue cysts of T. gondii in meat of infected livestock are an important source of infection for humans [10,11]

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