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

CORIGLIANO, MARIANA G. ; SÁNCHEZ-LÓPEZ, EDWIN F.; OLIFERUK, SONIA; RAMOS-DUARTE, VICTOR A.; RIVERA, MAXIMILIANO; MENDOZA-MORALES, LUISA F.; ANGEL, SERGIO O.; SANDER, VALERIA A.; CLEMENTE, MARINA

Frontiers in Plant Science

Frontiers Media S.A.

Año: 2021 vol. 12

le-interchange-newline">Plant 90 kDa heat shock protein (HSP90) is a potent adjuvant that increases both humoraland cellular immune responses to diverse proteins and peptides. In this study, we exploredwhether Arabidopsis thaliana HSP90 (AtHsp81.2) can improve the immune effects of aToxoplasma gondii surface antigen 1 (SAG1). We designed two constructs containingthe sequence of mature antigen (SAG1 m), from aa77 to aa322, and B- and T-cell antigenicepitope-containing SAG1 HC, from aa221 to aa319 fused to AtHsp81.2 sequence. Whencomparing the transient expression in Nicotiana tabacum X-27-8 leaves, which overexpressthe suppressor helper component protease HC-Pro-tobacco etch virus (TEV), to that inN. benthamiana leaves, co-agroinfitrated with the suppressor p19, optimal conditionsincluded 6-week-old N. benthamiana plants, 7-day time to harvest, Agrobacteriumtumefaciens cultures with an OD600nm of 0.6 for binary vectors and LED lights. WhileAtHsp81.2-SAG1 m fusion protein was undetectable by Western blot in any of the evaluatedconditions, AtHsp81.2?SAG1 HC was expressed as intact fusion protein, yielding up to90 μg/g of fresh weight. Besides, the AtHsp81.2?SAG1 HC mRNA was strongly expressedcompared to the endogenous Nicotiana tabacum elongation factor-alpha (NtEF α) gene,whereas the AtHsp81.2?SAG1 m mRNA was almost undetectable. Finally, mice were orallyimmunized with AtHsp81.2?SAG1 HC-infitrated fresh leaves (plAtHsp81.2?SAG1 HC group),recombinant AtHsp81.2?SAG1 HC purifid from infitrated leaves (rAtHsp81.2?SAG1 HCgroup), non-infitrated fresh leaves (control group), or phosphate-buffered saline (PBSgroup). Serum samples from plAtHsp81.2?SAG1 HC-immunized mice had signifiantlyhigher levels of IgGt, IgG2a, and IgG2b anti-SAG1 HC antibodies than serum fromrAtHsp81.2?SAG1 HC, control, and PBS groups. The number of cysts per brain in theplAtHsp81.2?SAG1 HC-immunized mice was signifiantly reduced, and the parasite load >in brain tissue was also lower in this group compared with the remaining groups. In animmunoblot assay, plant-expressed AtHsp81.2-SAG1 HC was shown to react withantibodies present in sera from T. gondii-infected people. Therefore, the plant expressionof a T. gondii antigen fused to the non-pathogenic adjuvant and carrier plant HSP90 asformulations against T. gondii can improve the vaccine effiacy, and plant extract canbe directly used for vaccination without the need to purify the protein, making this platforma suitable and powerful biotechnological system for immunogenic antigen expressionagainst toxoplasmosis.