Toxoplasma gondii is a zoonotic parasite that infects almost all warm-blooded animals and humans and results in serious health problems. There is no drug treatment for chronic toxoplasmosis. Thus, a safe and protective vaccine is required by the community to combat against the chronic infection caused by T. gondii in humans and animals. Rhoptry (ROP) proteins of T. gondii have important roles in host cell invasion, penetration, and biogenesis of the parasitophorous vacuole. In this study, we aimed to develop a novel vaccine with recombinant ROP6 protein (rROP6), which was shown to be highly immunogenic in protein microarray screening with blood samples collected from animal models infected with T. gondii oocysts or tissue cysts. Initially, a comprehensive in silico analyses was performed to design ROP6 protein to be used as vaccine antigen. Then, rROP6 protein was expressed in Saccharomyces cerevisiae INVSc1 cells and purified by affinity chromatography. Next, rROP6 protein adjuvanted with Freund's (rROP6 + Freund) was administered to BALB/c mice two times at three-week intervals. Humoral and cellular immune responses were analyzed by Western blot, ELISA, flow cytometry, and cytokine ELISA. Protective efficacy was determined by orally infecting mice with T. gondii PRU strain tissue cysts. The level of protection was analyzed by investigating tissue cysts in brain homogenate of mice using microscopy and qPCR. According to the results, rROP6 + Freund induced a strong IgG response compared to only rROP6 (P < 0.01) and the rate of CD8+ T lymphocytes secreting IFN- γ significantly increased in mice administered with rROP6 + Freund compared to other mice groups (P < 0.05). According to challenge results, all the rROP6 + Freund vaccine administered mice survived and the number of tissue cysts and the amount of T. gondii DNA decreased significantly compared to controls (P < 0.01; P < 0.0001). In conclusion, compared to control groups, rROP6 + Freund vaccine induced a high level of protective immunity and provided a significant level of protection as demonstrated by significant reduction in tissue cysts. We conclude that the recombinant ROP6 protein produced in S. cerevisiae is an immunogenic, protective, and promising vaccine candidate antigen that can be used in vaccine formulations against chronic toxoplasmosis.
