Abstract
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.
Highlights
Toxoplasmosis is caused by the parasite Toxoplasma gondii, an opportunistic pathogen that infects humans and warm-blooded animals (Peng et al, 2011)
The levels of anti-SAG1HC IgG1 elicited by immunization with plAtHsp81.2–SAG1HC were not significantly higher than those observed in the other groups (Figure 6A, Supplementary Figure S2). These results suggest that immunization with plAtHsp81.2–SAG1HC clearly shows a humoral immune response biased toward T helper 1 (Th1), associated with the production of IgG2a/IgG2b (Figure 6A, Supplementary Figure S2)
We demonstrated for the first time to our knowledge that a plant HSP90–SAG1HC fusion protein is expressed as intact protein at levels up to 90 μg/g of fresh weight (FW) when transiently co-expressed together with suppressor p19 in N. benthamiana and that oral vaccination with plant extracts containing AtHsp81.2-SAG1HC in mice elicits appropriate immune responses to prevent toxoplasmosis infection, reducing parasite cyst burden in brain tissue and symptoms of infection
Summary
Toxoplasmosis is caused by the parasite Toxoplasma gondii, an opportunistic pathogen that infects humans and warm-blooded animals (Peng et al, 2011). Congenital toxoplasmosis is the second most frequent intrauterine parasitic infection (Bojar and Szymańska, 2010); regular monitoring of pregnant women for this infection is mandatory (Oz, 2014) This parasite is geographically widely spread, and chronically infected animals are a permanent source of parasite transmission for humans (Kijlstra and Jongert, 2008; Tenter, 2009; Sander et al, 2018). The European Union has estimated that economic losses owing to abortions in these species are between 0.7 and 1.4 million euros per year (Katzer et al, 2011) For all these reasons, prevention of toxoplasmosis is a priority including through the development of effective vaccines (Pittman and Knoll, 2015)
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