PMID- 34675949 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20240508 IS - 1664-462X (Print) IS - 1664-462X (Electronic) IS - 1664-462X (Linking) VI - 12 DP - 2021 TI - 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. PG - 726910 LID - 10.3389/fpls.2021.726910 [doi] LID - 726910 AB - 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 Arabidopsis thaliana HSP90 (AtHsp81.2) can improve the immune effects of a Toxoplasma gondii surface antigen 1 (SAG1). We designed two constructs containing the sequence of mature antigen (SAG1(m)), from aa(77) to aa(322,) and B- and T-cell antigenic epitope-containing SAG1(HC), from aa(221) to aa(319) fused to AtHsp81.2 sequence. When comparing the transient expression in Nicotiana tabacum X-27-8 leaves, which overexpress the suppressor helper component protease HC-Pro-tobacco etch virus (TEV), to that in N. benthamiana leaves, co-agroinfiltrated with the suppressor p19, optimal conditions included 6-week-old N. benthamiana plants, 7-day time to harvest, Agrobacterium tumefaciens 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 90mug/g of fresh weight. Besides, the AtHsp81.2-SAG1(HC) mRNA was strongly expressed compared to the endogenous Nicotiana tabacum elongation factor-alpha (NtEFalpha) 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 T. gondii-infected people. Therefore, the plant expression of a T. gondii antigen fused to the non-pathogenic adjuvant and carrier plant HSP90 as formulations against T. gondii 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. CI - Copyright (c) 2021 Sanchez-Lopez, Corigliano, Oliferuk, Ramos-Duarte, Rivera, Mendoza-Morales, Angel, Sander and Clemente. FAU - Sanchez-Lopez, Edwin F AU - Sanchez-Lopez EF AD - Laboratorio de Molecular Farming y Vacunas, Instituto Tecnologico Chascomus (INTECH), Universidad Nacional de General San Martin (UNSAM), Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Chascomus, Argentina. FAU - Corigliano, Mariana G AU - Corigliano MG AD - Laboratorio de Molecular Farming y Vacunas, Instituto Tecnologico Chascomus (INTECH), Universidad Nacional de General San Martin (UNSAM), Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Chascomus, Argentina. FAU - Oliferuk, Sonia AU - Oliferuk S AD - Laboratorio de Molecular Farming y Vacunas, Instituto Tecnologico Chascomus (INTECH), Universidad Nacional de General San Martin (UNSAM), Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Chascomus, Argentina. FAU - Ramos-Duarte, Victor A AU - Ramos-Duarte VA AD - Laboratorio de Molecular Farming y Vacunas, Instituto Tecnologico Chascomus (INTECH), Universidad Nacional de General San Martin (UNSAM), Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Chascomus, Argentina. FAU - Rivera, Maximiliano AU - Rivera M AD - Laboratorio de Parasitologia Molecular, Instituto Tecnologico Chascomus (INTECH), Universidad Nacional de General San Martin (UNSAM), Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Chascomus, Argentina. FAU - Mendoza-Morales, Luisa F AU - Mendoza-Morales LF AD - Laboratorio de Molecular Farming y Vacunas, Instituto Tecnologico Chascomus (INTECH), Universidad Nacional de General San Martin (UNSAM), Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Chascomus, Argentina. FAU - Angel, Sergio O AU - Angel SO AD - Laboratorio de Parasitologia Molecular, Instituto Tecnologico Chascomus (INTECH), Universidad Nacional de General San Martin (UNSAM), Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Chascomus, Argentina. FAU - Sander, Valeria A AU - Sander VA AD - Laboratorio de Molecular Farming y Vacunas, Instituto Tecnologico Chascomus (INTECH), Universidad Nacional de General San Martin (UNSAM), Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Chascomus, Argentina. FAU - Clemente, Marina AU - Clemente M AD - Laboratorio de Molecular Farming y Vacunas, Instituto Tecnologico Chascomus (INTECH), Universidad Nacional de General San Martin (UNSAM), Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Chascomus, Argentina. LA - eng GR - R01 AI129807/AI/NIAID NIH HHS/United States PT - Journal Article DEP - 20211004 PL - Switzerland TA - Front Plant Sci JT - Frontiers in plant science JID - 101568200 PMC - PMC8525317 OTO - NOTNLM OT - Nicotiana benthamiana OT - SAG1 OT - adjuvant OT - fusion protein OT - heat shock protein OT - toxoplasmosis OT - vaccine COIS- The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. EDAT- 2021/10/23 06:00 MHDA- 2021/10/23 06:01 PMCR- 2021/01/01 CRDT- 2021/10/22 06:49 PHST- 2021/06/17 00:00 [received] PHST- 2021/08/30 00:00 [accepted] PHST- 2021/10/22 06:49 [entrez] PHST- 2021/10/23 06:00 [pubmed] PHST- 2021/10/23 06:01 [medline] PHST- 2021/01/01 00:00 [pmc-release] AID - 10.3389/fpls.2021.726910 [doi] PST - epublish SO - Front Plant Sci. 2021 Oct 4;12:726910. doi: 10.3389/fpls.2021.726910. eCollection 2021.