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

Abstract

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 <i>Arabidopsis thaliana</i> HSP90 (AtHsp81.2) can improve the immune effects of a <i>Toxoplasma gondii</i> surface antigen 1 (SAG1). We designed two constructs containing the sequence of mature antigen (SAG1_m), from aa₇₇ to aa_322, and B- and T-cell antigenic epitope-containing SAG1_HC, from aa₂₂₁ to aa₃₁₉ fused to AtHsp81.2 sequence. When comparing the transient expression in <i>Nicotiana tabacum</i> X-27-8 leaves, which overexpress the suppressor helper component protease HC-Pro-tobacco etch virus (TEV), to that in <i>N. benthamiana</i> leaves, co-agroinfiltrated with the suppressor p19, optimal conditions included 6-week-old <i>N. benthamiana</i> plants, 7-day time to harvest, <i>Agrobacterium tumefaciens</i> 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 <i>Nicotiana tabacum</i> 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 <i>T. gondii</i>-infected people. Therefore, the plant expression of a <i>T. gondii</i> antigen fused to the non-pathogenic adjuvant and carrier plant HSP90 as formulations against <i>T. gondii</i> 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.