Toxoplasma Gondii:Immunological Characterization Of Four Antioxidant & Glycolytic Enzymes

The intracellular protozoan parasite Toxoplasma gondii can infect a wide range of animal species. It is one of the main causes of infectious abortion and prenatal mortality in sheep. In humans, the parasite can cause abortion and congenital infection, and fatal disease in patients with immune incompetence. Attempts to control this disease in sheep were carried out by vaccination with a live attenuated vaccine; however, such vaccine has many disadvantages and is not acceptable for human application. Development of vaccines against T. gondii infection is of high demand, given the high burden of disease in some areas of the world like South America, and the lack of effective drugs with few adverse effects.Antioxidant enzymes as well as glycolytic enzymes play essential biological roles during the livelihood of T. gondii parasite. In this study we describe cloning and sequence analysis, prokaryotic expression and the immune potency based on DNA vaccines encoding selected antioxidant and glycolytic enzymes. The studies were carried out as follows:1-Cloning & sequence analysis of TgDPA, TgMDH, TgGR, and TgGST.Trizol reagent was used to extract the total RNA from T. gondii tachyzoites which were recovered from mice. Thereafter, mRNA was used to synthesize the cDNA, which was later applied as template during amplification of the required fragments of TgDPA, TgGR, TgGST and TgMDH. Specific primers for each gene were designed, with addition of enzyme restriction sites, based on the target sequences available in the GenBank. The PCR reaction was performed, and the amplified products were recovered and purified, followed by cloning into pMD-18T cloning vector and the ligation products were used to transform E. coli competent bacteria (DH5α). Restriction enzymes BamH I and Hind III were used to digest and identify positive clones with the right inserted DNA fragments and detection of the results took place by resolving the digestion products on 1% agarose gel. This step was later confirmed by submitting the recombinant products for further analysis by sequencing (Invitrogen, Shanghai) and bioinformatics analysis (DNASTAR Inc.). Bioinformatics analysis revealed the following;The ORF of TgDPA is 789bp and encodes a protein of 262 amino acids with molecular weight bout 27kDa. The ORF of TgMDH is 949bp and encodes a protein of 316 amino acids with molecular weight bout 34kDa. TgGR has an ORF of 1518bp and encodes a protein of 505 amino acids with molecular weight bout 54kDa. On the other hand, TgGST ORF contains 1056bp encoding a protein with 351 amino acids with a deduced protein about 39kDa..2-Prokaryotic expression of the selected genes and antisera DevelopmentThe recombinant proteins of TgDPA, TgGR, TgGST and TgMDH were obtained after the cloned DNA fragments were digested with the proper enzymes{BamH I and Hind III), recovered and sub-cloned into pET28a vector. Thereafter, the recombinant plasmids were used to transform E. coli (BL21) and expression of the recombinant proteins was induced with IPTG, and all the proteins were expressed as insoluble inclusion bodies. The expressed recombinant proteins were checked by running the bacterial extracts on SDS-PAGE gel, revealing bands about 27kDA for DPA,34kDa for MDH,54kDa for GR and 39kDa GST. After that, the recombinant proteins were passed through a scheme of dialysis and refolding processes. Antisera against rTgDPA, rTgGR, rTgGST and rTgMDH, were raised after rats were experimentally immunized with purified and re-natured recombinant proteins. Animals at the end of immunization session were bled and the sera were separated and western blot technique was used for verification. The recombinant proteins were immobilized on a membrane and probed with the anti-sera. Western blot revealed all the proteins had successfully stimulated the immune system of rats driving them to produce antibodies.3-Evaluation of the immune potency of TgDPA, TgGR, TgGST & TgMDHImmunological responses evaluated after vaccination with DNA vaccines constructed in this study, showed that specific humoral response defined by significant high titers of IgG (p<0.05) were induced only in immunized mice. In the groups vaccinated with TgDPA, TgGR, TgGST and TgMDH and compared to the controls, specific cytokines were analyzed (IFN-y, IL-4, IL-17 and TGF-β1). Levels of IFN-y, IL-4 and IL-17 demonstrated significant increase (p<0.05) following vaccination. TGF-β1 showed varied activity, no significant changes were detected in pTgGST group or in pTgMDH group, while in pTgDPA group decreased levels were recorded after the 2nd injection (p<0.05), meanwhile in pTgGR vaccinated group the production of TGF-β1 was significantly increased after the 2nd injection (p<0.05).The role of cell-mediated response has been well documented during vaccination trials against T. gondii infection. In this study, measurements of T lymphocyte subpopulations (CD4+ and CD8+) and MHC molecules were performed using flow cytometry. TgDPA vaccinated group as well as TgMDH vaccinated group, developed a significant response (p<0.05) resulted in the recruitment of both CD4+, CD8+ T cells subsets. In TgGR group cell-mediated response was dominated by CD4+T cell type (p<0.05), while TgGST vaccinated group showed a significant induction in CD8+T cell subset (P< 0.05).Experimental groups were challenged with T. gondii virulent strain RH (2*104) and survival rates were calculated. Mice in control groups died within 8 to 0 days after challenge, while vaccinated groups showed the following significant survival rates, TgDPA (20 days), TgMDH (17 days), TgGR (15 days) and TgGST (14 days). These results demonstrated that T. gondii antioxidant and glycolytic enzymes can induce significant humoral and cellular immune responses and produce partial protection against toxoplasmosis infection.