IntroductionToxoplasma gondii, an important intracellular parasite, causes toxoplasmosis which is a zoonotic disease results in serious health problems in humans and animals1. The life cycle of T. gondii is complex in which there is both sexual reproduction in definitive hosts (felids) and asexual reproduction in its intermediate hosts. T. gondii has three infective forms; the sporozoite occurs only in felids, the rapidly dividing tachyzoite occurs in definitive and intermediate hosts and is responsible for acute infection, and the slowly dividing bradyzoite form occurs in definitive and intermediate hosts and is responsible for chronic infection2. Acute infection sometimes causes headache, weakness, and fever in healthy people and chronic infection is usually asymptomatic which is represented by latent tissue cysts. Bradyzoites in these tissue cysts can reactivate and may cause death in immunocompromised individuals such as AIDS,cancer patients or organ transplant recipients3. Recently, tissue cysts located in the brain have been associated with behavioral disorders4. Humans as well as animals can become infected by consuming food and drinking water contaminated with oocysts containing sporozoites shed by cats or by eating meat contaminated with tissue cysts. Other transmission routes are transplacental transmission from mother to baby during pregnancy, organ transplantation or blood transfusion5.T. gondii infection can cause serious consequences in animals resulting with large scale economic losses especially in the small ruminant industry6. In the UK, toxoplasmosis is the second most prevalent cause of abortion in sheep7. Although T. gondii infection in pigs rarely causes clinical signs, several symptoms have been observed in sows, including abortion, fetal mummification, stillbirth, infertility, and neonatal mortality which results in financial losses8,9.Currently, several antiparasitic drugs are utilized for the treatment of acute toxoplasmosis in humans. However, no pharmacological agents have been identified as effective against chronic toxoplasmosis. The existing therapeutic options are associated with significant adverse effects, high toxicity, and the potential emergence of drug-resistant strains due to widespread use10. Furthermore, there is no approved vaccine that is both safe and effective for human use in preventing toxoplasmosis.To date, vaccine studies have been conducted using various platforms, new antigens, various expression systems, and new adjuvants11,12,13. Conventional vaccines based on attenuated or inactivated pathogens may contain genetic material from pathogen, there is a risk that they revert to a more virulent form in attenuated vaccines and moreover they have limited shelf life and stability, shorter duration of immunity and undesired immune response14,15. A live attenuated vaccine called ToxoVax® is used to prevent stillbirths in sheep, but there are some risks, such as conversion to a virulent strain16.On the other hand, subunit vaccines developed by biotechnological approaches do not carry the risk of reversion to virulent forms and are easily produced such as recombinant protein vaccines against HBV, HPV or SARS-CoV-217. Recombinant protein vaccine technology continues to develop promisingly as a safe and effective vaccine development platform against different pathogens. Recombinant protein vaccine studies are being carried out against toxoplasmosis using rhoptry proteins (ROP), microneme proteins (MIC), surface antigens (SAG) and dense granule antigens (GRA)12. Among them ROP proteins are secreted from rhoptry organelles of the parasite which are located at the apical end and have critical roles in parasite invasion, penetration, and biogenesis of parasitophorous vacuole. Moreover, they also ensure parasite survival by regulating host immune and metabolic responses18,19. In addition, ROP proteins which are synthesized in both tachyzoite and bradyzoite forms of the parasite were reported to be highly immunogenic20. In a study conducted by our study group, more than 2800 T. gondii exons were screened for their antigenicity using serum samples collected from mice experimentally infected with T. gondii oocysts and tissue cysts as well as humans with toxoplasmosis and the results revealed that ROP6 protein was highly immunogenic21,22,23. Transcriptomic and proteomic analyses of T. gondii confirmed that ROP6 protein expression level is elevated in sporozoites at the beginning of infection suggesting an important role in host cell invasion24,25. Recombinant proteins can be expressed in various expression systems and yeast expression systems are frequently used due to their post-translational modification capability, suitability for large-scale production, and non-pathogenicity. Saccharomyces cerevisiae is a Generally Recognized as Safe (GRAS) and is recognized as the safest microorganism by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA)11,14,26,27. In this study, the rROP6 protein was expressed for the first time in Saccharomyces cerevisiae INVSc1 cells, purified, and adjuvanted with Freund’s adjuvant. Subsequently, rROP6 + Freund vaccine was administered to BALB/c mice and immunogenicity was analyzed by Western blot, IgG ELISA, cytokine ELISA, and flow cytometry. Protective efficacy was determined by investigating tissue cysts in brain homogenates of vaccinated mice orally infected with tissue cysts of T. gondii PRU strain using microscopy and qPCR.ResultsIn silico analysis of ROP6 proteinIn silico analysis showed that the native full length ROP6 protein (1443 bp, 480 aa, 51.4 kDa) was not stable and insoluble (Supplementary file) (Table 1). A previous study showed that full length ROP6 protein aggregates when expressed in E. coli due to the residues between 421 and 480 aa28. Therefore, we decided to truncate the ROP6 protein to make it more stable. Accordingly, transmembrane (455–477 aa) and intracellular domains (478–480 aa) identified using the TMHMM 2.0 program were removed, and the remaining domain (454 aa) was analyzed for the presence of T and B cell epitopes as well as glycosylation sites. In the remaining region, seven T (five MHC-I and two MHC-II) and five B cell epitopes were determined by IEDB and SVMTrip programs and moreover two N-glycosylation and two O-glycosylation sites were identified using NetNGlyc 1.0 and NetOGlyc 4.0 programs (Supplementary file, S1 Table and S2 Table). Based on the ProtParam stability scores, epitope and glycosylation sites, ROP6 protein between 2 and 156 aa was used as an antigen that contained 3 B cell and 6 T cell (five MHC-I and one MHC-II) epitopes (Supplementary file, S2 Table). To increase the stability of truncated ROP6 protein, serine amino acid belonging to S. cerevisiae Kozak sequence (AAAAAAATGTCC)29 and valine amino acid were added to the N-terminal right after methionine and Kozak sequence (MSV) (Supplementary file, S1 table)30. The ORF in the resulting pYES2.1/ROP6 vector expressed a truncated recombinant ROP6 protein with a size of 208 aa (theoretical molecular weight 22.15 kDa) was stable (Supplementary file, S1 Table) (Table 1).Table 1 Physico-chemicals properties of full length ROP6 and designed ROP6 proteins.Full size tablerROP6 protein expression in S. cerevisiae and purificationS. cerevisiae INVSc1 cells that expressed rROP6 protein were grown in SC-U induction medium and disrupted with microfluidizer processor for purification. After high-speed centrifuge, the supernatant was filtered and rROP6 protein was purified by AKTA-FPLC chromatography system using a HiTrap Ni2 + chelating column. Purified rROP6 protein is shown in Fig. 1. Although the theoretical molecular weight of rROP6 was 22.15 kDa, it appeared as ~ 27 kDa possibly due to post translational modifications in S. cerevisiae. There were some more bands in SDS-PAGE and Western blot band which may be due to multimerization or degradation of rROP6 protein.Fig. 1(A) SDS-PAGE and (B) Western blot images of rROP6 protein purified by HiTrap Ni2+ chelating column. Lane 1: Protein ladder; Lane 2: rROP6 protein (~27 kDa). Red arrow heads show the rROP6 protein.Full size imageHumoral immune responseAnti-rROP6 IgG antibody response induced by vaccination was analyzed by Western blot and ELISA. In Western blot, rROP6 containing membranes were probed with sera pools prepared from mice administered with rROP6 + Freund and control groups mice. The results showed the presence of anti-rROP6 IgG antibodies in sera of mice administered with rROP6 + Freund vaccine. No bands were detected in sera of control group mice (Fig. 2A2A). Furthermore, rROP6 containing membranes were probed with T. gondii PRU strain infected and negative control mice serum samples. The results showed the presence of anti-rROP6 IgG antibodies in sera of naturally T. gondii infected mice. No bands were detected in sera of negative control group mice (Fig. 2B). In another Western blot, TLA containing membranes were probed with sera pools prepared from mice administered with rROP6 + Freund and control groups mice, the results showed that anti-rROP6 IgG antibodies produced by rROP6 + Freund vaccine specifically recognized the native ROP6 protein, exhibiting a molecular weight of ~ 51.4 kDa (Fig. 2 C).Fig. 2(A) Western blot analysis using recombinant ROP6 (rROP6) protein as the antigen revealed the presence of anti-rROP6 IgG antibodies in the sera of mice immunized with rROP6 formulated with Freund’s adjuvant, collected two weeks after the final vaccination (day 35). Lane 1: Protein ladder; Lane 2: sera of mice administered with PBS; Lane 3: sera of mice administered with Freund; Lane 4: sera of mice administered with rROP6; Lane 5: sera of mice administered with rROP6+Freund; Lane 6: rROP6 protein probed with anti-poly his antibody. Red arrow heads show rROP6 protein with a size of ~27 kDa (B) Western blot analysis using rROP6 protein as the antigen revealed the presence of anti-rROP6 IgG antibodies in the sera of mice infected with T. gondii PRU strain tissue cysts Lane 1: Protein ladder; Lane 2: sera of negative control mice; Lane 3-4: sera of mice infected with T. gondii PRUstrain tissue cysts. Red arrow heads show rROP6 protein with a size of ~27 kDa (C) Western blot analysis using T. gondii lysate antigen (TLA) as the coating antigen demonstrated that anti-rROP6 IgG antibodies present in the sera of mice immunized with rROP6+Freund (collected on day 35, two weeks after the final immunization) specifically recognized the native ROP6 protein, exhibiting a molecular weight of ~51.4 kDa. Lane 1: Protein ladder; Lane 2: sera of mice administered with PBS; Lane 3: sera of mice administered with Freund; Lane 4: sera of mice administered with rROP6; Lane 5: sera of mice administered with rROP6+Freund; Red arrow heads show native T. gondii ROP6 protein (D) ELISA results showing anti-rROP6 IgG responses obtained from mice group vaccinated with rROP6+Freund, only rROP6, Freund and PBS collected before vaccination and two weeks after last vaccination (day 35) **: P