AUTHOR=Sánchez-López Edwin F. , Corigliano Mariana G. , Oliferuk Sonia , Ramos-Duarte Victor A. , Rivera Maximiliano , Mendoza-Morales Luisa F. , Angel Sergio O. , Sander Valeria A. , Clemente Marina 

TITLE=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

JOURNAL=Frontiers in Plant Science

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.726910

DOI=10.3389/fpls.2021.726910

ISSN=1664-462X

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