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

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.

Histone variant H2B.Z acetylation is necessary for maintenance ofToxoplasma gondiibiological fitness

“…It is relevant that these tachyzoites were able to generate a complete immune response, which only happens when cells are invaded by tachyzoites and not with an infection with dead or lysed tachyzoites [85][86][87][88]. In this sense, Toxovac® is a live attenuated vaccine used to prevent sheep congenital toxoplasmosis [89].…”

“…Western blot was then performed as described [90]. The primary antibodies: αrH2B.Z [22], αrH2A.Z [23], αrROP5 (rabbit) and αrROP18 (rabbit) were used at 1/5000, whereas α-c-Myc (rabbit, abCam ab9106) was used at 1/2000 and αrSag1 [87] 1/200 for 1 h at room temperature. αH3ac (rabbit, Millipore, 06-599B) 1/200, was incubated overnight.…”

“…Samples were boiled for 5 min and 20 µl were loaded per well in a 15% SDS-PAGE gel for immunoblotting. The absence of contaminating proteins was corroborated by Western blot with murine anti-SAG1antibody [87]. Quantification of the bands was done with Image J 1.53q (Fiji) software.…”

Safe plant Hsp90 adjuvants elicit an effective immune response against SARS-CoV2 derived RBD antigen

“…However, it is more laborious, and the expression levels of the recombinant proteins vary from case to case. In this case, it was also shown to be highly efficient in generating an adequate immune response[7,13,17].…”

Recombinant production of SAG1 fused with xylanase in Pichia pastoris induced higher protective immunity against Eimeria tenella infection in chicken

“…It has been used as a potential target for immune response and shown to provide protection against Eimeria infection as DNA or subunit vaccines (Pagheh et al, 2020). Enhancements to its immune protective capacity have been achieved through fusion or co-administration with certain proteins including IL-2, IL-18, HSP90, et al (Liu et al, 2010;Sanchez-Lopez et al, 2021;Song et al, 2009;Xu et al, 2008). In addition, multiple antigen delivery systems, including PLGA nanoparticle, Salmonella typhimurium, Lactobacillus plantarum, improve its protective effect (Allahyari et al, 2022;Liu et al, 2020;Pogonka et al, 2003).…”