These sequences do not contain the N-terminal signal peptide or the C-terminal hydrophobic region (Supplementary Figure S1). orally immunized with AtHsp81.2CSAG1HC-infiltrated fresh leaves (plAtHsp81.2CSAG1HC group), recombinant AtHsp81.2CSAG1HC purified from infiltrated leaves (rAtHsp81.2CSAG1HC group), non-infiltrated fresh leaves (control group), or phosphate-buffered saline (PBS group). Serum samples from plAtHsp81.2CSAG1HC-immunized mice had significantly higher levels of IgGt, IgG2a, and IgG2b anti-SAG1HC antibodies than serum from rAtHsp81.2CSAG1HC, control, and PBS groups. The number of cysts per brain in the plAtHsp81. 2CSAG1HC-immunized mice was significantly reduced, and the parasite load in brain tissue PHCCC 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 antigen fused to the non-pathogenic adjuvant and carrier plant HSP90 as formulations against 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. is an efficient parasite, affecting one in three people (Pittman and Knoll, 2015). Those with the highest risk of developing serious infections are pregnant women, children with congenital infections, and immunocompromised patients (Petersen et al., 2010). Congenital toxoplasmosis is the second most frequent intrauterine parasitic infection (Bojar and Szymaska, 2010); therefore, regular monitoring of pregnant women for this infection is mandatory (Oz, 2014). Furthermore, 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). In fact, the Center for Disease Control (CDC) in the United States includes in a list of neglected parasitic infections.1 While the cat is the definitive host, mammals and birds are intermediate hosts. infection in livestock is an important source of economic losses owing to resultant spontaneous abortions and neonatal deaths, especially in sheep and goats (Dubey et al., 1998). 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). Protein-based subunits vaccines are generally considered safe; however, they can be weakly immunogenic, often requiring the addition of adjuvants to elicit an effective immune response (Perez et al., PHCCC 2012; Jo et al., 2021). Despite several adjuvants have been assayed in experimental subunit vaccines against (Sander et al., 2018; ?ahar et al., 2020), the search for safer and non-toxic adjuvants capable of strongly boosting and directing immune responses is still one of the main challenges in this field. Several plant molecules have shown adjuvant capacity, including saponins, polysaccharides, and lectins (Sander et al., 2018). Rabbit Polyclonal to TLK1 In addition, in the last decade, heat shock proteins (HSPs) have emerged as new potent plant adjuvants (Buriani et al., 2011, 2012; Corigliano et al., 2013; Bengoa-Luoni et al., 2019; Snchez-Lpez et al., 2019). HSP90s have been proposed as danger signals since they are inducible proteins whose synthesis increases under certain stress situations such as heat, cold, irradiation, or presence of some pathogens such as viruses and bacteria, and even bacterial toxins (Moseley, 1998). Therefore, when HSP90s are released into the extracellular environment due to cell damage or necrosis, they can bind to surface receptors present on professional antigen-presenting cells (pAPCs) and trigger a pro-inflammatory immune response, thereby modulating the innate immune response (Binder et al., 2000). In particular, this ability occurs, at least, through interaction with TLR2 and TLR4 receptors (Rico et al., 2002). Since both foreign and endogenous HSP90s are involved in the presentation of antigens to pAPCs and PHCCC in the maturation of these cells, the potential role of HSP90s as adjuvants was recognized favoring antigen presentation (Suzue and Young, 1996; More PHCCC et al., 1999; Basu et al., 2001; Echeverria et al., 2006; Tobian et al., 2014; Corigliano et al., 2021). Corigliano et al. (2011) showed that plant cytosolic HSP90s, Hsp90.3 (NbHsp90.3) and Hsp81.2 (AtHsp81.2), stimulate the proliferation of B cells and that TLR4 is involved in this mitogenic interaction. In.