Construction of A Synthetic Gene Encoding the Multi-Epitope of Toxoplasma gondii and Demonstration of the Relevant Recombinant Protein Production: A Vaccine Candidate

Abstract

Background: Toxoplasma gondii is a widely-distributed parasite all over the world whose attributed severe afflicting complications in human necessitate the development of serodiagnostic tests and vaccines for it. Immunological responses to monovalent vaccines and the application of diagnostic reagents including single antigens are not optimally effective. Bioinformatics approaches were used to introduce these epitopes, predict their immunogenicity and preliminarily evaluate their potential as an effective DNA vaccine and for serodiagnostic goals. Materials and Methods: A 3D structure of proteins was predicted by I-TASSER server, and linear and conformational B cell and T cell epitopes were predicted using the online servers. Then, the predicted epitopes were constructed and called Toxoeb, and their expression in the prokaryotic and eukaryotic cells was demonstrated using SDS-PAGE. In the next step, Western blotting with pooled sera of mice infected with T. gondii was done. Results: The current in silico analysis revealed that the B cell epitopes with high immunogenicity for GRA4 protein were located in the residues 34-71, and 230-266, for GRA14 in 308-387, for SAG1 in 182-195, 261-278, and for GRA7 in residues 101-120, 160-176. The T cell epitopes were selected in overlapping regions with the B cell epitopes. The immunogenic region for GRA4 are in the residues 245-253, 50-58, and 40-54, for GRA14 in 307-315, 351-359, and 308-322, for SAG1 261-269, and 259-267, and for GRA7 in the residues 103-112, and 167-175. The results of the western blotting showed that the expressed protein had immunogenicity. Conclusion: Our constructed multi-epitope of T. gondii could be considered as a candidate for diagnostic and vaccination purposes. [GMJ.2020;9:e1708]

Toxoplasma gondii vaccine candidates: a concise review

“…Accordingly, significant advances have been made in characterization and isolation of antigens, gene cloning, antigen expression, and immunological methods. In addition to the prevention strategies, new options are now needed to develop effective vaccines as a way to prevent the toxoplasmosis [ 29 ]. Most of the T. gondii antigens are important for the virulence and immunogenicity of the parasite.…”

“…Recombinant antigen vaccines have advantages over classic methods. One of the problems with using live vaccines is that if the host’s immune system is defective, it may cause the tachyzoite to return from an attenuated form to an active invasive form [ 29 ]. Another disadvantage is the complexity of obtaining sufficient amounts of purified immunogenic components of the antigen by the classical methods.…”

“…GRA7 is expressed in all of the stages of T. gondii infection and is a considerable candidate for the vaccine design [ 91 , 93 ]. It can effectively induce both cellular and humoral immune responses against the T. gondii [ 29 ]. Another member of this family is GRA4 that is a 40 KDa protein secreted into the parasitophorous vacuole by the parasite [ 40 , 41 ].…”

“…The GRA4 stimulates the mucosal T lymphocytes in BALB/C and CBA/J mice strains. The GRA4 can induce mucosal and systemic immune responses in mice after T. gondii ingestion [ 24 , 29 , 41 ]. Furthermore, GRA14 is a 47 KDa protein with 409 amino acids.…”

“…In people with a healthy immune system, the symptoms of the infection usually are similar to mild flu symptoms, while in immunocompromised patients, it can cause severe and even life-threatening complication such as encephalitis and severe ocular complications. In addition, in pregnant women, toxoplasmosis may cause abortion or congenital toxoplasmosis with manifestations such as neurological or ocular in the fetus [ 6 , 29 ]. It is estimated that approximately 50% of untreated maternal infections are transmitted to the fetus in which approximately 60% are subclinical, 30% have severe damage such as hydrocephalus, intracerebral calcification, retinochoroiditis (Classical triad) and mental retardation and 9% are fatal for the fetus [ 30 ].…”