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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  Arabidopsis thaliana  HSP90 (AtHsp81.2) can improve the immune effects of a  Toxoplasma gondii  surface antigen 1 (SAG1). We designed two constructs containing the sequence of mature antigen (SAG1 m ), from aa 77  to aa 322,  and B- and T-cell antigenic epitope-containing SAG1 HC , from aa 221  to aa 319  fused to AtHsp81.2 sequence. When comparing the transient expression in  Nicotiana tabacum  X-27-8 leaves, which overexpress the suppressor helper component protease HC-Pro-tobacco etch virus (TEV), to that in  N. benthamiana  leaves, co-agroinfiltrated with the suppressor p19, optimal conditions included 6-week-old  N. benthamiana  plants, 7-day time to harvest,  Agrobacterium tumefaciens  cultures with an OD 600nm  of 0.6 for binary vectors and LED lights. While AtHsp81.2-SAG1 m  fusion protein was undetectable by Western blot in any of the evaluated conditions, AtHsp81.2–SAG1 HC  was expressed as intact fusion protein, yielding up to 90μg/g of fresh weight. Besides, the AtHsp81.2–SAG1 HC  mRNA was strongly expressed compared to the endogenous  Nicotiana tabacum  elongation factor-alpha (NtEFα) gene, whereas the AtHsp81.2–SAG1 m  mRNA was almost undetectable. Finally, mice were orally immunized with AtHsp81.2–SAG1 HC -infiltrated fresh leaves (plAtHsp81.2–SAG1 HC  group), recombinant AtHsp81.2–SAG1 HC  purified from infiltrated leaves (rAtHsp81.2–SAG1 HC  group), non-infiltrated fresh leaves (control group), or phosphate-buffered saline (PBS group). Serum samples from plAtHsp81.2–SAG1 HC -immunized mice had significantly higher levels of IgGt, IgG2a, and IgG2b anti-SAG1 HC  antibodies than serum from rAtHsp81.2–SAG1 HC , control, and PBS groups. The number of cysts per brain in the plAtHsp81.2–SAG1 HC -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 HC  was shown to react with antibodies present in sera from  T. gondii -infected people. Therefore, the plant expression of a  T. gondii  antigen fused to the non-pathogenic adjuvant and carrier plant HSP90 as formulations against  T. gondii  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.

frontiers in plant science

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