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

SANCHEZ LOPEZ E; CORIGLIANO M; OLIFERUK S; RAMOS-DUARTE VA; RIVERA M; MENDOZA-MORALES LF; ANGEL SO; SANDER VA; CLEMENTE M

Frontiers in Plant Science

Frontiers Media S.A.

Año: 2021 vol. 12 p. 2070 - 2089

Plant 90 kDa heat shock protein (HSP90) is a potent adjuvant that increases both humoral26 and cellular immune responses to diverse proteins and peptides. In this study, we explored27 the immune effects of a Toxoplasma gondii surface antigen 1 (SAG1)?HSP90. We28 designed two constructs containing the sequence of mature antigen (SAG1m), from aa7729 to aa322, and B- and T-cell antigenic epitope-containing SAG1HC, from aa221 to aa319 fused30 to Arabidopsis thaliana HSP90 (AtHsp81.2). The optimal conditions for the expression31 of AtHsp81.2-SAG1HC included the use of p19 suppressor, 6-week-old plants, 7-days32 time to harvest, Agrobacterium tumefaciens cultures with an OD600nm of 0.6 for binary33 vectors and LED lights. Agroinfiltrations by vacuum or syringe methods with the34 constructs were carried out either on N. tabacum X-27-8 leaves or on N. benthamiana35 leaves co-agroinfiltrated with the suppressor p19. While AtHsp81.2-SAG1m fusion36 protein was undetectable by Western blot in any of the evaluated conditions, AtHsp81.2?37 SAG1HC was expressed as intact fusion protein, yielding up to 90 μg/g of fresh weight.38 Besides, the AtHsp81.2?SAG1HC mRNA was strongly expressed compared to the39 endogenous Nicotiana tabacum elongation factor-alpha (NtEFα) gene, whereas the40 AtHsp81.2?SAG1m mRNA was almost undetectable. Finally, mice were orally41 immunized with AtHsp81.2?SAG1HC-infiltrated fresh leaves (plAtHsp81.2?SAG1HC),42 recombinant AtHsp81.2?SAG1HC purified from infiltrated leaves (rAtHsp81.2?43 SAG1HC), non-infiltrated fresh leaves (control), or phosphate buffered saline (PBS).44 Serum samples from plAtHsp81.2?SAG1HC-immunized mice had significantly higher45 levels of IgGt, IgG2a and IgG2b anti-rSAG1m antibodies than serum from rAtHsp81.2?46 SAG1HC, control and PBS groups. The number of cysts per brain in the plAtHsp81.2?47 SAG1HC-immunized mice was significantly reduced, and the parasite load in brain tissue48 was also lower in this group compared with the remaining groups. In an immunoblot49 assay, plant-expressed AtHsp81.2-SAG1HC was shown to react with antibodies present in 350 sera from T. gondii-infected people. Therefore, the plant expression of a T. gondii antigen51 fused to the non-pathogenic adjuvant and carrier plant HSP90 as formulations against T.52 gondii can improve the vaccine efficacy, making this platform a suitable and powerful biotechnological system for immunogenic antigen expression against toxoplasmosis.