Assessment of an in-house ELISA and IgG avidity test based on SAG1 and GRA7 proteins for discriminating between acute and chronic toxoplasmosis in humans

To improve serodiagnostic methods for diagnosis of acute from chronic toxoplasmosis, an economical in-house ELISA for measuring Toxoplasma -specific IgG, IgM and IgG avidity has been developed and assessed based on use of various T. gondii antigens, including SAG1, GRA7 and a combination of SAG1 and GRA7 (SAG1+GRA7) as well as Toxoplasma lysate antigens (TLAs). Performances of in-house IgM, IgG and IgG avidity assays were compared to those of ELISA commercial kits and VIDAS Toxo IgG avidity. A set of 138 sera from patients with acquired T. gondii infection and seronegative people were assessed. Receiver operating characteristic (ROC) analysis revealed an area under curve (AUC) of 0.98, 0.97, 0.99 and 0.99 for IgM-TLAs, IgM-SAG1, IgM-GRA7, and IgM-SAG1+GRA7, respectively. Furthermore, AUC was calculated as 0.99, 0.99, 0.98 and 0.99 for IgG-TLAs, IgG-SAG1, IgG-GRA7 and IgG-SAG1+GRA7, respectively. The current study showed that GRA7 included 100% sensitivity for the detection of Toxo IgM, while SAG1 included 89.7% sensitivity. Furthermore, the highest specificity (97.2%) to detect Toxo IgM was achieved using SAG1+GRA7 antigen. For the detection of Toxo IgG, the highest sensitivity (100%) was recorded for SAG1+GRA7 followed by TLAs (97.9%). The SAG1+GRA7 showed the greatest potential for assessing avidity of IgG antibodies with 97.1% of sensitivity and 96.6% of specificity, compared to VIDAS Toxo IgG avidity. The preliminary results have promised better discriminations between acute and chronic infections using a combination of SAG1 and GRA7 recombinant antigens, compared to TLAs.

Evaluation of Toll-Like Receptor 11 Agonist Adjuvant Activity in Immunization of BALB/c Mice with Total Lysate Antigens of Toxoplasma gondii RH Strain

Background: In this study, the effect of total lysate antigen (TLA) of Toxoplasma gondii on spleen lymphocyte prolifration, secretion of IL5, INF-γ, and mice survival time was evaluated using agonist of toll-like receptor (TLR) 11, as an adjuvant. Results: Mice immunized with TLA + adjuvant showed higher immunization index than the two other groups and combination of TLR11 (as an adjuvant) and TLA significantly elevated the effect of TLA by increasing the production of INF-γ and IL-5 and by the shift of the immune system to Th1. In addition, the combination of TLA and TLR11 adjuvant increased the proliferation of lymphocytes and survival time in mice against T. gondii. Conclusion: Profilin (as an adjuvant) in combination with TLA could be a potent vaccine candidate that evokes a powerful specific immune response and significantly improves the efficacy of TLA vaccine by increasing the induction of INF-γ production and by shifting the immune responses to Th1 profile through increasing the INF-γ/IL-5 ratio. It causes significant protection against T. gondii after i.p. injection.

Recombinant Dense Granular Protein (GRA5) for Detection of Human Toxoplasmosis by Western Blot

Toxoplasma gondiiinfects all warm-blooded animals, including humans, causing serious public health problems and great economic loss for the food industry. Commonly used serological tests require costly and hazardous preparation of wholeToxoplasmalysate antigens from tachyzoites. Here, we have evaluated an alternative method for antigen production, which involved a prokaryotic expression system. Specifically, we expressedT. gondiidense granular protein-5 (GRA5) inEscherichia coliand isolated it by affinity purification. The serodiagnostic potential of the purified recombinant GRA5 (rGRA5) was tested through Western blot analysis against 212 human patient serum samples. We found that rGRA5 protein was 100% specific for analysis of toxoplasmosis-negative human sera. Also, rGRA5 was able to detect acute and chronicT. gondiiinfections (sensitivities of 46.8% and 61.2%, resp.).