Production of the main surface antigen of Toxoplasma gondii in tobacco leaves and analysis of its antigenicity and immunogenicity

We adapted a previously described Agrobacterium-mediated transient expression system to test the expression level of three constructs carrying the surface antigen 1 (SAG1) of Toxoplasma gondii. Two constructs were based in a Potato virus X (PVX) amplicon. In one of them, the PVX movement protein genes were replaced by the sag1 gene. In the other, the sag1 gene was placed under the control of an additional coat protein subgenomic promoter. In the third construct, the sag1 gene was fused to an apoplastic peptide signal under the CaMV 35S promoter. Western blot analysis of leaf extracts infiltrated with each construct revealed a protein of 35 kDa. SAG1 accumulation in leaves ranged from 0.1 to 0.06% of total soluble protein (equivalent to 10 μg and 6 μg of SAG1 per gram of fresh leaf tissue, respectively). Three of five human seropositive samples reacted with tobacco-expressed SAG1 in Western blot analysis. The C3H mice were immunized with SAG-expressing leaf extracts and perorally challenged with a nonlethal dose of the T. gondii Me49 strain. Mice vaccinated with SAG1 showed significantly lower brain cyst burdens compared to those from the control group. Immunization with SAG1-expressing leaves elicited a specific humoral response with predominant participation of type IgG2a. In conclusion, a functional SAG1 version could be transiently expressed in tobacco leaves.