Study On Immunoprotection From Toxoplasmosis Provided By A WH3-GRA15-GFP Nuclei Acid Vaccine

Background:Toxoplasma gondii is a specially designed cell in parasitic protozoa and parasitic in the human and many animals in eukaryotic cell, causing zoonotic toxoplasmosis. Most infected people have no clinical symptoms and signs, but in those with the human immunodeficiency virus it can cause serious consequences. There is no effective drug for prevention and treatment of toxoplasmosis, thus vaccine prevention is the direction of development. At present, Toxoplasma gondii vaccine study has not given rise to a practical and effective candidate miolecule which can be used. The reason for the unsuccessfulness may be the complex life cycle of the parasite, with specific antigens of diversification, high degree of variability, multiple invasion pathways, and evasion of the host immune attack mechanism. With the development of molecular biology and gene engineering, a progress has been made in the research of T.gondii vaccine development. Here we used WH3-GRA15-GFP nucleic acid vaccine by intramuscular immunization of mice and detected the immunogenicity and efficacy of immune protection so as to screen the candidate vaccines against Toxoplasma infection.Objective:To construct the recombinant eukaryotic expression plasmids of Toxoplasma GRA15, and to assess the immune protection in BALB/C mice induced by a WH3-GRA15-GFP nuclei acid vaccination.Methods:T. gondii GRA15 coding gene obtained by PCR amplification was inserted into the eukaryotic expression vector pEGFP-C2 to generate recombinant WH3-GRA15-GFP. T293 cells were transfected by Liposome method in vitro and the fusion protein expression was identified by Western blotting. Fifty-four BALB/C mice were randomly divided into experimental group, empty plasmid group, and control group, with 18 mice in each.The mice were immunized via injection in the quadriceps femoris.The experimental group was injected with 100μg of the recombinant plasmid WH3-GRA15-GFP, the empty plasmid group was injected with 100μg of the pEGFP-C2 plasmid, and the PBS group was injected with 100μL of PBS buffer. Each immunization was done a total of three times with an interval of 2 weeks. Before each injection and two weeks after the final injection, levels of serum IgG, IgG1, and IgG2a antibodies and cytokines(IL-2、IL-4、IL-10、IFN-γ) were measured. Thirteen mice in all of the groups were challenged intraperitoneslly with 1,000 tachyzoites of the strain of T. gondii two weeks after the final injection to observe their survial time and evaluate immunity.Results:WH3-GRA15-GFP nucleic acid was found to be expressed in mice but failed to induce effective cellular and humoral immune response. Compared with control group,both the levels of the specific antibodies (IgG, IgG1, IgG2a) and cytokines (IL-2, IL-4,IL-10, and IFN-γ) and the number of deaths of mice did not reveal a statistically significant change (P>0.05).Conclusions:Dense granular protein of GRA15 was not effectively enough to serve as Toxoplasma vaccine candidate molecule. The GRA15 prediction protein is not stable and has less antigen epitopes coupled with a relatively simple 3D structure.WH3-GRA15-GFP DNA vaccine of T.gondii may not be an effective vaccine candidate molecule due to its failure to induce immune protection in BALB/c mice.