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

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

Frontiers in Plant Science

Frontiers

Lugar: Lausanne; Año: 2021 vol. 12

Plant 90kDa heat shock protein (HSP90) is a potent adjuvant thatincreases both humoral and cellular immune responses to diverseproteins and peptides. In this study, we explored whether Arabidopsisthaliana HSP90 (AtHsp81.2) can improve the immune effects of aToxoplasma gondii surface antigen 1 (SAG1). We designed twoconstructs containing the sequence of mature antigen (SAG1m), fromaa77 to aa322, and B- and T-cell antigenic epitope-containing SAG1HC,from aa221 to aa319 fused to AtHsp81.2 sequence. When comparing thetransient expression in Nicotiana tabacum X-27-8 leaves, whichoverexpress the suppressor helper component protease HC-Pro-tobaccoetch virus (TEV), to that in N. benthamiana leaves,co-agroinfiltrated with the suppressor p19, optimal conditionsincluded 6-week-old N. benthamiana plants, 7-day time to harvest,Agrobacterium tumefaciens cultures with an OD600nm of 0.6 for binaryvectors and LED lights. While AtHsp81.2-SAG1m fusion protein wasundetectable by Western blot in any of the evaluated conditions,AtHsp81.2?SAG1HC was expressed as intact fusion protein, yieldingup to 90μg/g of fresh weight. Besides, the AtHsp81.2?SAG1HC mRNAwas strongly expressed compared to the endogenous Nicotiana tabacumelongation factor-alpha (NtEFα) gene, whereas the AtHsp81.2?SAG1mmRNA was almost undetectable. Finally, mice were orally immunizedwith AtHsp81.2?SAG1HC-infiltrated fresh leaves (plAtHsp81.2?SAG1HCgroup), recombinant AtHsp81.2?SAG1HC purified from infiltratedleaves (rAtHsp81.2?SAG1HC group), non-infiltrated fresh leaves(control group), or phosphate-buffered saline (PBS group). Serumsamples from plAtHsp81.2?SAG1HC-immunized mice had significantlyhigher levels of IgGt, IgG2a, and IgG2b anti-SAG1HC antibodies thanserum from rAtHsp81.2?SAG1HC, control, and PBS groups. The numberof cysts per brain in the plAtHsp81.2?SAG1HC-immunized mice wassignificantly reduced, and the parasite load in brain tissue was alsolower in this group compared with the remaining groups. In animmunoblot assay, plant-expressed AtHsp81.2-SAG1HC was shown to reactwith antibodies present in sera from T. gondii-infected people.Therefore, the plant expression of a T. gondii antigen fused to thenon-pathogenic adjuvant and carrier plant HSP90 as formulationsagainst T. gondii can improve the vaccine efficacy, and plant extractcan be directly used for vaccination without the need to purify theprotein, making this platform a suitable and powerfulbiotechnological system for immunogenic antigen expression againsttoxoplasmosis.p { margin-bottom: 0.25cm; line-height: 120%; background: transparent }