Development Of Synthetic Multiepitope Peptide (Rmep) As Potential Serodiagnostic Marker And Vaccine Candidate For Toxoplasma gondii Infection
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Date
2016-05
Authors
Haj Issa Idrees, Khalid Mohamed Ali
Journal Title
Journal ISSN
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Publisher
Universiti Sains Malaysia
Abstract
Infection with the intracellular parasite Toxoplasma gondii exhibits a worldwide distribution. Such disease is known to cause abortions and serious clinical complications on the fetus, neonate, and immunocompromised individuals, resulting in considerable clinical and economic effects. The most effective measure for controlling toxoplasmosis and minimizing the harms caused by the parasite is prompt diagnosis and treatment. Meanwhile, vaccination is an efficient tool for preventing the occurrence of the infection. Therefore, development of a novel antigen for diagnostic or vaccination purposes is important.
Significant efforts have been made to acquire such antigen. As a result, developing multi-epitope-based antigens using software-based prediction tools and molecular techniques may provide a novel and alternative means for acquiring less expensive and more accurate diagnostic kits or potential vaccine candidates. The advantage of the multi-epitope antigen lies in the capacity to combine epitopes from different stages of the parasite. Thereby, this approach would serve as a promising and valuable strategy to overcome the antigen complexity of the T. gondii life cycle.
In this study, a single synthetic gene of approximately 456 bp in size, which encodes potential epitopes of T. gondii antigens, was successfully constructed using gene assembly PCR. The constructed gene, designated as USM.TOXO1, was then cloned into a pET32a(+) expression vector and transformed into BL21 (DE3) pLysS E. coli Competent cells. The entire protein was successfully expressed and purified. Subsequently, the immunoreactivity of this antigen was evaluated by developing immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using human sera. Meanwhile, the immunogenicity was tested in BALB/c mice.
The usefulness of USM.TOXO1 for the diagnosis of toxoplasmosis through ELISA was tested on 151 sera from positive T. gondii patients and 96 sera from negative patients for the detection of specific anti-T. gondii IgG. The USM.TOXO1 ELISA presented an 85.43% sensitivity, 81.25% specificity, 87.76% positive predictive value, and 78% negative predictive value.
Immunization of the BALB/c mice with USM.TOXO1 generated a strong mixed Th1/Th2 response polarized toward the IgG1 antibody isotype. Additionally, analysis of cytokine profiles following in vitro stimulation revealed the significant synthesis of IFN-γ cytokines, but not IL-4, in the immunized mice compared with the control group.
In conclusion, USM.TOXO1 is a potential serodiagnostic marker for the detection of T. gondii infection in humans, as well as a promising vaccine candidate that elicits protective immunity in BALB/c mice. The proven
immunoreactivity and immunogenicity of USM.TOXO1 can serve as a premise for the further use of epitope-based antigens in the routine diagnosis and immunoprevention of human and animal toxoplasmosis.
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Keywords
Vaccination is an efficient tool for , preventing the occurrence of the infection.