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.

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 (SAG1m), from aa77 to aa322, and B- and T-cell antigenic epitope-containing SAG1HC, from aa221 to aa319 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 OD600nm of 0.6 for binary vectors and LED lights. While AtHsp81.2-SAG1m fusion protein was undetectable by Western blot in any of the evaluated conditions, AtHsp81.2-SAG1HC was expressed as intact fusion protein, yielding up to 90µg/g of fresh weight. Besides, the AtHsp81.2-SAG1HC mRNA was strongly expressed compared to the endogenous Nicotiana tabacum elongation factor-alpha (NtEFα) gene, whereas the AtHsp81.2-SAG1m mRNA was almost undetectable. Finally, mice were orally immunized with AtHsp81.2-SAG1HC-infiltrated fresh leaves (plAtHsp81.2-SAG1HC group), recombinant AtHsp81.2-SAG1HC purified from infiltrated leaves (rAtHsp81.2-SAG1HC group), non-infiltrated fresh leaves (control group), or phosphate-buffered saline (PBS group). Serum samples from plAtHsp81.2-SAG1HC-immunized mice had significantly higher levels of IgGt, IgG2a, and IgG2b anti-SAG1HC antibodies than serum from rAtHsp81.2-SAG1HC, control, and PBS groups. The number of cysts per brain in the plAtHsp81.2-SAG1HC-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-SAG1HC 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.

Use of Veterinary Vaccines for Livestock as a Strategy to Control Foodborne Parasitic Diseases.

Foodborne diseases (FBDs) are a major concern worldwide since they are associated with high mortality and morbidity in the human population. Among the causative agents of FBDs, Taenia solium, Echinococcus granulosus, Toxoplasma gondii, Cryptosporidium spp., and Trichinella spiralis are listed in the top global risk ranking of foodborne parasites. One common feature between them is that they affect domestic livestock, encompassing an enormous risk to global food production and human health from farm to fork, infecting animals, and people either directly or indirectly. Several approaches have been employed to control FBDs caused by parasites, including veterinary vaccines for livestock. Veterinary vaccines against foodborne parasites not only improve the animal health by controlling animal infections but also contribute to increase public health by controlling an important source of FBDs. In the present review, we discuss the advances in the development of veterinary vaccines for domestic livestock as a strategy to control foodborne parasitic diseases.

Heat Shock Proteins 90 kDa: Immunomodulators and Adjuvants in Vaccine Design Against Infectious Diseases.

Heat shock proteins 90 kDa (Hsp90s) were originally identified as stress-responsive proteins and described to participate in several homeostatic processes. Additionally, extracellular Hsp90s have the ability to bind to surface receptors and activate cellular functions related to immune response (cytokine secretion, cell maturation, and antigen presentation), making them very attractive to be studied as immunomodulators. In this context, Hsp90s are proposed as new adjuvants in the design of novel vaccine formulations that require the induction of a cell-mediated immune response to prevent infectious diseases. In this review, we summarized the adjuvant properties of Hsp90s when they are either alone, complexed, or fused to a peptide to add light to the knowledge of Hsp90s as carriers and adjuvants in the design of vaccines against infectious diseases. Besides, we also discuss the mechanisms by which Hsp90s activate and modulate professional antigen-presenting cells.

Heat treatment alleviates the growth and photosynthetic impairment of transplastomic plants expressing Leishmania infantum Hsp83-Toxoplasma gondii SAG1 fusion protein.

Previously, we showed that transplastomic tobacco plants expressing the LiHsp83-SAG1 fusion protein displayed a chlorotic phenotype and growth retardation, while plants expressing the SAG1 and GRA4 antigens alone did not. We conducted a comprehensive examination of the metabolic and photosynthetic parameters that could be affecting the normal growth of LiHsp83-SAG1 plants in order to understand the origin of these pleiotropic effects. These plants presented all photosynthetic pigments and parameters related to PSII efficiency significantly diminished. However, the expression of CHLI, RSSU and LHCa/b genes did not show significant differences between LiHsp83-SAG1 and control plants. Total protein, starch, and soluble sugar contents were also greatly reduced in LiHsp83-SAG1 plants. Since Hsp90 s are constitutively expressed at much higher concentrations at high temperatures, we tested if the fitness of LiHsp83-SAG1 over-expressing LiHsp83 would improve after heat treatment. LiHsp83-SAG1 plants showed an important alleviation of their phenotype and an evident recovery of the PSII function. As far as we know, this is the first report where it is demonstrated that a transplastomic line performs much better at higher temperatures. Finally, we detected that LiHsp83-SAG1 protein could be binding to key photosynthesis-related proteins at 37 °C. Our results suggest that the excess of this molecular chaperone could benefit the plant in a possible heat shock and prevent the expected denaturation of proteins. However, the LiHsp83-SAG1 protein content was weakly decreased in heat-treated plants. Therefore, we cannot rule out that the alleviation observed at 37 °C may be partially due to a reduction of the levels of the recombinant protein.

Plant Serine Protease Inhibitors: Biotechnology Application in Agriculture and Molecular Farming.

The serine protease inhibitors (SPIs) are widely distributed in living organisms like bacteria, fungi, plants, and humans. The main function of SPIs as protease enzymes is to regulate the proteolytic activity. In plants, most of the studies of SPIs have been focused on their physiological role. The initial studies carried out in plants showed that SPIs participate in the regulation of endogenous proteolytic processes, as the regulation of proteases in seeds. Besides, it was observed that SPIs also participate in the regulation of cell death during plant development and senescence. On the other hand, plant SPIs have an important role in plant defense against pests and phytopathogenic microorganisms. In the last 20 years, several transgenic plants over-expressing SPIs have been produced and tested in order to achieve the increase of the resistance against pathogenic insects. Finally, in molecular farming, SPIs have been employed to minimize the proteolysis of recombinant proteins expressed in plants. The present review discusses the potential biotechnological applications of plant SPIs in the agriculture field.

Plant Hsp90 is a novel adjuvant that elicits a strong humoral and cellular immune response against B- and T-cell epitopes of a Toxoplasma gondii SAG1 peptide.

BACKGROUND: The 90-kDa heat-shock protein (Hsp90) from Nicotiana benthamiana (NbHsp90.3) is a promising adjuvant, especially for those vaccines that require a T cell-mediated immune response. Toxoplasma gondii SAG1 is considered one of the most important antigens for the development of effective subunit vaccines. Some epitopes located in the SAG1 C-terminus region have showed a strong humoral and cellular immune response. In the present study, we aimed to assess the efficacy of NbHsp90.3 as carrier/adjuvant of SAG1-derived peptide (SAG1HC) in a T. gondii infection murine model. METHODS: In the present study, C57BL/6 mice were intraperitoneal immunized with the NbHsp90.3-SAG1HC fusion protein (NbHsp90.3-SAG1HC group), mature SAG1 (SAG1m group), NbHsp90.3 (NbHsp90.3 group) or PBS buffer 1× (PBS group). The levels of IgG antibodies and the cytokine profile were determined by ELISA. Two weeks after the last immunization, all mice were orally challenged with 20 cysts of T. gondii Me49 strain and the number of brain cysts was determined. In addition, both humoral and cellular immune responses were also evaluated during the acute and chronic phase of T. gondii infection by ELISA. RESULTS: The characterization of the immune response generated after vaccination with NbHsp90.3 as an adjuvant showed that NbHsp90.3-SAG1HC-immunized mice produced antibodies that were able to recognize not only rSAG1m but also the native SAG1 present in the total lysate antigen extract (SAG1TLA) from T. gondii tachyzoites, while control groups did not. Furthermore, anti-rSAG1m IgG2a/2b antibodies were significantly induced. In addition, only the spleen cell cultures from NbHsp90.3-SAG1HC-immunized mice showed a significantly increased production of IFN-γ. During the chronic phase of T. gondii infection, the antibodies generated by the infection were unable to detect the recombinant protein, but they did react with TLA extract. In addition, splenocytes from all groups showed a high production of IFN-γ when stimulated with rGRA4, but only those from NbHsp90.3-SAG1HC group stimulated with rSAG1m showed high production of IFN-γ. Finally, NbHsp90.3-SAG1HC-immunized mice exhibited a significant reduction in the cyst load (56%) against T. gondii infection. CONCLUSIONS: We demonstrated that NbHsp90.3 enhances the humoral and cell-mediated immune response through a Th1 type cytokine production. Mice vaccinated with NbHsp90.3-SAG1HC exhibited a partial protection against T. gondii infection and it was correlated with the induction of memory immune response. We developed and validated a vaccine formulation which, to our knowledge, for the first time includes the NbHsp90.3 protein covalently fused to a peptide from T. gondii SAG1 protein that contains T- and B-cell epitopes.