Oral Immunization With a Plant HSP90-SAG1 Fusion Protein Produced in Tobacco Elicits Strong Immune Responses and Reduces Cyst Number and Clinical Signs of Toxoplasmosis in Mice.

Plant 90kDa heat shock protein (HSP90) is a potent adjuvant that increases both humoral and cellular immune responses to diverse proteins and peptides. In this study, we explored whether HSP90 (AtHsp81.2) can improve the immune effects of a surface antigen 1 (SAG1). We designed two constructs containing the sequence of mature antigen (SAG1), from aa to aa and B- and T-cell antigenic epitope-containing SAG1, from aa to aa fused to AtHsp81.2 sequence. When comparing the transient expression in X-27-8 leaves, which overexpress the suppressor helper component protease HC-Pro-tobacco etch virus (TEV), to that in leaves, co-agroinfiltrated with the suppressor p19, optimal conditions included 6-week-old plants, 7-day time to harvest, cultures with an OD of 0.6 for binary vectors and LED lights. While AtHsp81.2-SAG1 fusion protein was undetectable by Western blot in any of the evaluated conditions, AtHsp81.2-SAG1 was expressed as intact fusion protein, yielding up to 90μg/g of fresh weight. Besides, the AtHsp81.2-SAG1 mRNA was strongly expressed compared to the endogenous elongation factor-alpha (NtEFα) gene, whereas the AtHsp81.2-SAG1 mRNA was almost undetectable. Finally, mice were orally immunized with AtHsp81.2-SAG1-infiltrated fresh leaves (plAtHsp81.2-SAG1 group), recombinant AtHsp81.2-SAG1 purified from infiltrated leaves (rAtHsp81.2-SAG1 group), non-infiltrated fresh leaves (control group), or phosphate-buffered saline (PBS group). Serum samples from plAtHsp81.2-SAG1-immunized mice had significantly higher levels of IgGt, IgG2a, and IgG2b anti-SAG1 antibodies than serum from rAtHsp81.2-SAG1, control, and PBS groups. The number of cysts per brain in the plAtHsp81.2-SAG1-immunized mice was significantly reduced, and the parasite load in brain tissue was also lower in this group compared with the remaining groups. In an immunoblot assay, plant-expressed AtHsp81.2-SAG1 was shown to react with antibodies present in sera from -infected people. Therefore, the plant expression of a antigen fused to the non-pathogenic adjuvant and carrier plant HSP90 as formulations against can improve the vaccine efficacy, and plant extract can be directly used for vaccination without the need to purify the protein, making this platform a suitable and powerful biotechnological system for immunogenic antigen expression against toxoplasmosis.