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 whetherArabidopsis thalianaHSP90 (AtHsp81.2) can improve the immune effects of aToxoplasma gondiisurface antigen 1 (SAG1). We designed two constructs containing the sequence of mature antigen (SAG1m), from aa77to aa322,and B- and T-cell antigenic epitope-containing SAG1HC, from aa221to aa319fused to AtHsp81.2 sequence. When comparing the transient expression inNicotiana tabacumX-27-8 leaves, which overexpress the suppressor helper component protease HC-Pro-tobacco etch virus (TEV), to that inN. benthamianaleaves, co-agroinfiltrated with the suppressor p19, optimal conditions included 6-week-oldN. benthamianaplants, 7-day time to harvest,Agrobacterium tumefacienscultures with an OD600nmof 0.6 for binary vectors and LED lights. While AtHsp81.2-SAG1mfusion protein was undetectable by Western blot in any of the evaluated conditions, AtHsp81.2–SAG1HCwas expressed as intact fusion protein, yielding up to 90μg/g of fresh weight. Besides, the AtHsp81.2–SAG1HCmRNA was strongly expressed compared to the endogenousNicotiana tabacumelongation factor-alpha (NtEFα) gene, whereas the AtHsp81.2–SAG1mmRNA was almost undetectable. Finally, mice were orally immunized with AtHsp81.2–SAG1HC-infiltrated fresh leaves (plAtHsp81.2–SAG1HCgroup), recombinant AtHsp81.2–SAG1HCpurified from infiltrated leaves (rAtHsp81.2–SAG1HCgroup), non-infiltrated fresh leaves (control group), or phosphate-buffered saline (PBS group). Serum samples from plAtHsp81.2–SAG1HC-immunized mice had significantly higher levels of IgGt, IgG2a, and IgG2b anti-SAG1HCantibodies than serum from rAtHsp81.2–SAG1HC, control, and PBS groups. The number of cysts per brain in the plAtHsp81.2–SAG1HC-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-SAG1HCwas shown to react with antibodies present in sera fromT. gondii-infected people. Therefore, the plant expression of aT. gondiiantigen fused to the non-pathogenic adjuvant and carrier plant HSP90 as formulations againstT. gondiican 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.