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Toxoplasma gondii is an obligate intracellular parasite that causes one of the most common parasitic infections in humans and other warm-blooded animals. Currently, there are no effective treatments for inhibiting the formation of chronic tissue cysts in infected hosts. Thus, the development of a vaccine to protect against toxoplasmosis is an attractive option for avoiding infection. The aim of this study was to design an epitope-based vaccine for T. gondii. In the present study, an in silico approach was used to predict and analyze B-cell and T-cell epitopes and the transmembrane domain of proteins SAG1, MIC3, and ROP8. We also predicted the antigenicity, allergenicity, secondary and tertiary structures, and physicochemical characteristics of a chimeric protein. Next, codon optimization and mRNA structure prediction were conducted using bioinformatics tools, and the designed construct was chemically synthesized and cloned into the pET28a vector. SAG1 (amino acid positions 85–235), MIC3 (30–180), and ROP8 (85–185) were found to have several strong immunodominant epitopes that were joined with a rigid linker A(EAAAK)2A. Although the resultant protein called MRS ( M IC3, R OP8, and S AG1) did not turn out to be an allergen, its antigenicity was estimated to be 0.7983. Additionally, MRS was selected as the best vaccine candidate on the basis of its secondary and tertiary structures. The number of amino acids, molecular weight, and numbers of negatively and positively charged residues of MRS were 427 and 45,661.31 Da, 45, and 50, respectively. ΔG of the best-predicted structure was −413.0 kcal/mol, and the first nucleotides at the 5′ end did not form a stable hairpin or pseudoknot. Finally, successful expression and verification of the expressed MRS protein showed that in silico analysis was almost accurate. This vaccine candidate selected by in silico tools should be validated in experimental studies.

Toxoplasma gondii is an intracellular apicomplexan parasite, which can cause a serious infectious disease in pregnant women and immunocompromised individuals. Therefore, the development of a polyvalent vaccine consisting of all stages of the parasite life cycle using the epitopes from tachyzoites, bradyzoites, and sporozoites is likely to be required for complete protective immunity. In this study, we designed protein vaccine candidate based on the prediction of specific epitopes (i.e., B cell and T cell) from three Toxoplasma gondii antigens. The MRS protein (MIC3: 30–180, ROP8: 85–185, and SAG1: 85–235) was expressed in Escherichia coli, and purification was performed using a HisTrap HP column and then we evaluated immunogenicity and protective property in BALB/c mice. Seventy-two mice were randomly divided into six groups, including three vaccinations (i.e., MRS, MRS-Freund, and MRS-Calcium Phosphate Nanoparticles (MRS-CaPNs)) and three control (i.e., Phosphate-buffered saline, Freund, and CaPNs) groups. All groups were immunized three times via subcutaneous injection within three-week intervals. In the vaccination groups, the BALB/c mice were injected with 20 μg of MRS protein for the first time and 10 μg of MRS for the next two times. Antibodies, cytokines, and splenocytes proliferation in the immunized mice were assayed using the enzyme-linked immunosorbent assay. Protective efficacy was analyzed by challenging the immunized mice with T. gondii of RH strain. Antibody, cytokine, and lymphocyte proliferation assays showed that the mice immunized with MRS induced stronger humoral and T helper type 1 cell-mediated immune responses, compared to the control mice. However, co-immunization with adjuvants (i.e., Freund and CaNPs) resulted in impaired immune responses. Effective protection against the parasite achieved an increase in survival time in the immunized mice, especially in the MRS-CaNPs group. The obtained results of the present study demonstrated that multi-epitope protein vaccination, MRS, is a potential strategy against toxoplasmosis infection. In addition, the vaccine co-delivered with CaPNs could provide an important key for vaccine candidate to control T. gondii infection.

Toxoplasma gondii, the etiological agent of toxoplasmosis, can cause severe or lethal damages in both animals and man. So, tends to develop a more effective vaccine to prevent this disease is extremely needed and would be so prominent. The novel dense granule antigen 14 (GRA14) has been identified as a potential vaccine candidate against T. gondii infection. The aim of this study was evaluation of protective immunity induced by prime/boost vaccination strategy of GRA14 antigen with calcium phosphate (CaPNs) or Aluminum hydroxide (Alum) nano-adjuvants in BALB/c mice. The finding showed that immunization with the prime-boost strategy using plasmid DNA (pcGRA14) and recombinant protein (rGRA14) with nano-adjuvants significantly elicited levels of specific IgG antibodies and cytokines against T. gondii infection. Given that, there were the high levels of total IgG, IgG2a, IFN-γ in mice of rGRA14-CaPNs and pcGRA14 + rGRA14-CaPNs groups, which indicating a Th-1 type response. While immunization of mice with Alum based rGRA14 and pcGRA14 + rGRA14 elicited specific IgG1 and IL-4 levels, which was confirmed a Th-2 type response. Mice immunized with DNA prime-protein boost vaccine with nano-adjuvants produce more vigorous specific lymphoproliferative responses than mice immunized with other antigen formulations. In addition, the CaPNs-based prime-boost vaccine of pcGRA14 + rGRA14 showed the longest survival time in mice and the lowest parasitic load in their brain tissue compared to the other groups. The results obtained in this study show that the use of GRA14 based DNA prime–protein boost vaccination regime with CaPNs can dramatically enhanced both humoral and cellular immune responses. Therefore, this strategy can provide a promising approach to the development of an effective vaccine against T. gondii infection in the future.