Toxoplasmosis is an important zoonotic disease caused by Toxoplasma gondii that can infect almost all warm-blooded animals worldwide, including humans. The high prevalence of T. gondii infection and its ability to cause serious harm to humans and animals, especially immunodeficient individuals, make it a key public health issue. Accurate diagnostic tools with high sensitivity are needed for controlling T. gondii infection. In the current study, we compared the performance of recombinant SAG2, GRA6, and GRA7 in ELISA for the serological diagnosis of T. gondii infection in cats. We further investigated the antigenicity of recombinant dense granule protein 3 (rGRA3), rGRA5, rGRA8, and rSRS29A expressed in a plant-based, cell-free expression system for detecting antibodies in T. gondii-infected cats. In summary, our data suggest that GRA7 is more sensitive than the other two antigens for the serodiagnosis of T. gondii infection in cats, and GRA3 expressed in the cell-free system is also a priming antigen in serological tests for detecting T. gondii infection in cats.

Toxoplasmosis is a zoonotic disease caused by the protozoan parasite, Toxoplasma gondii known to infect almost all animals, including birds and humans globally. This disease has impacted the livestock industry and public health, where infection of domestic animals increases the zoonotic risk of transmission of infection to humans, threatening public health. Hence the need to discover novel and safe vaccines to fight against toxoplasmosis. In the current study, a novel multiepitope vaccine was designed using immunoinformatics techniques targeting T. gondii AMA1, GRA7 and ROP16 antigens, consisting of antigenic, immunogenic, non-allergenic and cytokine inducing T-cell (9 CD8+ and 15 CD4+) epitopes and four (4) B-cell epitopes fused together using AAY, KK and GPGPG linkers. The tertiary model of the proposed vaccine was predicted and validated to confirm the structural quality of the vaccine. The designed vaccine was highly antigenic (antigenicity = 0.6645), immunogenic (score = 2.89998), with molecular weight of 73.35 kDa, instability and aliphatic index of 28.70 and 64.10, respectively; and GRAVY of -0.363. The binding interaction, stability and flexibility were assessed with molecular docking and dynamics simulation, which revealed the proposed vaccine to have good structural interaction (binding affinity = -106.882 kcal/mol) and stability when docked with Toll like receptor-4 (TLR4). The results revealed that the Profilin-adjuvanted vaccine is promising, as it predicted induction of enhanced immune responses through the production of cytokines and antibodies critical in blocking host invasion.

Toxoplasma gondii is an apicomplexan parasite that affects the health of humans and livestock, and an effective vaccine is urgently required. Nanoparticles can modulate and improve cellular and humoral immune responses.
In the current study, poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles were used as a delivery system for the T. gondii dense granule antigens GRA12 and GRA7. BALB/c mice were injected with the vaccines and protective efficacy was evaluated.
Mice immunized with PLGA+GRA12 exhibited significantly higher IgG, and a noticeable predominance of IgG2a over IgG1 was also observed. There was a 1.5-fold higher level of lymphocyte proliferation in PLGA+GRA12-injected mice compared to Alum+GRA12-immunized mice. Higher levels of IFN-g and IL-10 and a lower level of IL-4 were detected, indicating that Th1 and Th2 immune responses were induced but the predominant response was Th1. There were no significant differences between Alum+GRA7-immunized and PLGA+GRA7-immunized groups. Immunization with these four vaccines resulted in significantly reduced parasite loads, but they were lowest in PLGA+GRA12-immunized mice. The survival times of mice immunized with PLGA+GRA12 were also significantly longer than those of mice in the other vaccinated groups.
The current study indicated that T. gondii GRA12 recombinant protein encapsulated in PLGA nanoparticles is a promising vaccine against acute toxoplasmosis, but PLGA is almost useless for enhancing the immune response induced by T. gondii GRA7 recombinant protein.

Toxoplasmosis is a zoonotic disease caused by the obligate intracellular protozoan parasite T. gondii which is widely prevalent in humans and animals worldwide. The diagnosis of toxoplasmosis and distinguishing acute or chronic T. gondii infections have utmost importance for humans and animals. The TgSAG1, TgGRA7, and TgBAG1 proteins were used in the present study to develop the serological rSAG1-ELISA, rGRA7-ELISA and rBAG1-ELISA methods for the testing of T. gondii specific IgG and IgM antibodies and differentiating acute or chronic toxoplasmosis in 3733 animals, including Tibetan sheep, yaks, pigs, cows, cattle, horses, chickens, camels and donkeys from the Qinghai-Tibetan Plateau. The ELISA tests showed that the overall positivity of IgG antibody was 21.1% (786/3733), 15.3% (570/3733) and 18.2% (680/3733) for rSAG1-, rGRA7- and rBAG1-ELISA, respectively, and the positivity of IgM antibody was 11.8% (439/3733), 13.0% (486/3733) and 11.8% (442/3733) for rSAG1-, rGRA7- and rBAG1-ELISA, respectively. A total of 241 animals (6.5%) positive for all rSAG1-, rGRA7- and rBAG1-IgG were found in this study, and the 141 animals (3.8%) tested were anti-T. gondii IgM positive in all three ELISAs. Moreover, the 338, 284 and 377 animals were IgG positive in rSAG1 + rGRA7-, rBAG1 + rGRA7- and rSAG1 + rBAG1- ELISAs respectively, and the 346, 178 and 166 animals in rSAG1 + rGRA7-, rBAG1 + rGRA7- and rSAG1 + rBAG1-ELISAs were IgM positive respectively. The results confirmed that the application of SAG1, GRA7, and BAG1 recombinant antigens could successfully be used in the detection of specific IgG and IgM antibodies for distinguishing between acute or chronic T. gondii infections. It is inferred that the forms in which current animal species in the plateau area were infected with T. gondii, and the period of infection or the clinical manifestations of the current infections may be different. The present study provides substantial clinical evidence for the differential diagnosis of toxoplasmosis, and the classification of acute and chronic T. gondii infections.

UNASSIGNED: Toxoplasma gondii is recognized as a zoonosis causing toxoplasmosis in animals globally. Cat is a definitive host of T. gondii and sheds oocyst through feces, which can infect human beings and animals through contaminated food ingestion. A precise diagnostic test is essential to prevent T. gondii infection in both humans and animals. This study aimed to develop and evaluate the pETite-dense granule antigen 7(GRA7)-based indirect enzyme-linked immunosorbent assay (ELISA) to detect T. gondii infection in cats.
UNASSIGNED: T. gondii-GRA7 was cloned and expressed in the Expresso®small ubiquitin-related modifier (SUMO) T7 Cloning and Expression System. The recombinant pETite-GRA7 was purified using HisTrap affinity chromatography and confirmed using Western blot analysis. The recombinant protein was used to develop and evaluate the indirect ELISA for T. gondii infection detection. In total, 200 cat sera were tested using pETite-GRA7-based indirect ELISA and indirect fluorescent antibody test (IFAT). The statistical analysis based on Kappa value, sensitivity, specificity, positive predictive value, negative predictive value, χ 2 test, and receiver operating characteristic (ROC) curve was used to evaluate the performance of the test.
UNASSIGNED: A 606 bp GRA7 polymerase chain reaction (PCR) product was obtained from T. gondii RH strain genomic DNA. The gene was cloned into the pETite™ vector and transformed to HI-Control Escherichia coli BL21 (DE3) for protein expression. Approximately 35 kDa of recombinant pETite-GRA7 was observed and Western blot analysis showed positive bands against anti-6-His antibody and positive-T. gondii cat serum. A sample of 0.5 μg/mL of pETite-GRA7 was subjected to indirect ELISA to detect T. gondii infection in the cat sera. The results showed sensitivity and specificity of pETite-GRA7-based indirect ELISA at 72% and 96%, respectively. An acceptable diagnostic performance was characterized by high concordant results (94%) and substantial agreement (Kappa value=0.65) with IFAT. The seroprevalence levels of ELISA and IFAT were 10% and 9%, respectively, and were not significantly (p>0.05) different. The expected performance of ELISA at different cutoff points using the ROC curve analysis revealed 89% sensitivity and 92% specificity at the cutoff value of 0.146, with a high overall assay accuracy (area under the curve=0.94).
UNASSIGNED: In this study, the pETite™ vector, N-terminal 6xHis SUMO fusion tag, was used to improve the solubility and expression level of GRA7. The recombinant pETite-GRA7 showed enhanced protein solubility and purification without special condition requirements. This pETite-GRA7-based indirect ELISA showed high concordant results and substantial agreement with IFAT. ELISA revealed an acceptable sensitivity and specificity. These initial data obtained from cats\' sera demonstrated that pETite-GRA7-based indirect ELISA could be a useful method for local serological diagnosis of T. gondii infection in cats in Thailand.

BACKGROUND: Discovery of new Toxoplasma gondii serotyping epitopes is important due to reports showing the influence of genotype on the severity of toxoplasmosis. In Turkey, genotypes belonging to type II, type III and Africa 1 lineages were mainly detected. The present study focused on to find out epitopes with high discriminative capacity to serotype these genotypes using well characterized strains isolated from Turkey.
METHODS: To meet this objective, GRA6 and GRA7 genes were sequenced from strains belonging to the type II, III and Africa 1 lineages, and B cell epitopes inside these sequences were predicted by Bcepred and additional docking analysis was performed with B cell receptor. Based on these analyses, 22 peptides harboring lineage specific epitopes were synthesized. Then, the serotyping potency of these peptides was tested using peptide ELISA and well categorized serum samples collected from stray cats infected with genotypes of the different lineages type II (n:9), III (n:1) and Africa 1 (n:1). As a result of peptide-ELISA, a serotyping schema was constructed with peptides that show high discriminative capacity and this assay was validated by sera collected from humans after an outbreak (n:30) and mother/newborn pair sera (n:3). Later, the validated serotyping schema was used to serotype a larger group of human (n:38) and cat (n:24) sera.
RESULTS: Among 22 peptides, GRA6II/c, GRA7III/d, and GRA6 Africa 1/b epitopes have shown discriminative capacity. During the validation of peptide-ELISA, the serotype of toxoplasmosis outbreak and mother/newborn cases were detected to be serotype II. Moreover, the analyses in a larger group showed that serotype II was prevalent in humans and stray cats.
CONCLUSIONS: Overall, the results showed that the serotyping schema could be successfully used to serotype T. gondii infections caused by type II, III and Africa 1 genotype.

To improve serodiagnostic methods for diagnosis of acute from chronic toxoplasmosis, an economical in-house enzyme-linked immunosorbent assay (ELISA) for measuring Toxoplasma-specific IgG, IgM, and IgG avidity has been developed and assessed based on use of various Toxoplasma 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% sensitivity and 96.6% specificity compared to those of 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 those using TLAs.

Toxoplasmosis is a zoonotic disease with worldwide prevalence in humans and warm-blooded animal populations. In livestock Toxoplasma gondii is the causal agent of significant economic losses since it can cause abortions in goats and sheep. It is estimated that one third of the world population is infected. Although there are effective therapies for acute infection, these are sometimes poorly tolerated, teratogenic, and have a long administration time. Considering the deficiencies that exist related to the prevention and treatment of toxoplasmosis, the development of a safe and effective vaccine would be extremely valuable in fighting against this infection. In the present work, we characterize for the first time the adjuvant and immunogenic potential of a recombinant profilin protein (rTgPF), in a vaccine formulation alone or in combination with the well-known GRA7 antigen candidate in a murine toxoplasmosis model. Since TgPF acts as a ligand for TLR11 and 12 inducing innate immune responses that promote type 1 adaptive responses, we first study the capacity of the mix rGRA7 + rTgPF to initiate an immune response by evaluating dendritic cell activation. Both rTgPF and rGRA7 induces activation of mouse BMDCs more efficiently than the single proteins, evidenced by increased expression of CD80 and CD86 co-stimulatory proteins and secretion of IL-6, IL-10 and IL-12 cytokines after in vitro stimulation. The sum of the effects of rGRA7 and rTgPF on BMDCs maturation led us to assay them in a vaccination protocol. BALB/c mice vaccinated with this mix elicited a Th1-biased immunity via the induction of lymphocyte proliferation, activation of CD4+T cells and increased IFN-γ production that resulted in enhanced protection against chronic Toxoplama gondii infection. Profilin per se induce only cellular immunity but augments the effect of rGRA7 immune responses when used together, thus allowing us to postulate rTgPF as a potential adjuvant in a protein vaccine.

Toxoplasmosis, caused by Toxoplasma gondii, is one of the most common parasitic diseases worldwide. The GRA7 of T. gondii (TgGRA7) is an essential component of the parasitophorous vacuole (PV) and PV membrane surrounding the tachyzoites and the cyst wall of the bradyzoites. While it has been widely employed as antigen for ELISA, there is only one study that has reported its potential as antigen for immunochromatographic test (ICT) in pigs. There is no study yet documenting its potential for ICT serodiagnosis of T. gondii infection in cats. In this study, we assessed the efficacy of an ICT using TgGRA7 in the detection of Toxoplasma infections in 100 cats and compared the results with iELISAs using TgGRA7 and lysate antigens of T. gondii strains, RH, PLK, and VEG. Our results revealed that TgGRA7-ICT is a reliable test for the diagnosis of anti-T. gondii antibodies in cats, producing comparable results as conventional serological methods. This study is the first report on the use of TgGRA7 as ICT antigen for the serodiagnosis of T. gondii infection in cats.

Toxoplasma gondii variant influences clinical profile in human congenital and ocular toxoplasmosis. Parasite genotyping represents a challenge due to insufficient amount of genetic material of the protozoan in the host samples, and isolates are hard to obtain, especially from pediatric patients. An alternative is serotyping, which is based on the presence of specific antibodies against polymorphic proteins related to virulence; the more widely used are GRA6 and GRA7, but most works report cross reactions among the classical strains (I, II, and III). We designed new peptides of GRA6, GRA7, and SAG1 proteins, with more SNPs among the three clonal strains than those previously designed. This was done by identifying BcR and polymorphic epitopes by means of bioinformatics; then we designed peptides with linkers joining the specific regions and predicted their 3D structure. With the commercial molecules synthesized on the basis of these designs, we tested 86 serum samples from 42 mother/newborn pairs and two congenitally infected newborns, by indirect ELISA. We implemented a strategy to determine the serotype based on scatter plots and a mathematical formula, using ratios among reactivity indexes to peptides. We found low frequency of samples reactive to GRA7 and SAG1, and cross reactions between GRA6 serotypes I and III; we modified these later peptides and largely improved distinction among the three clonal strains. The chronicity of the infection negatively affected the reactivity index against the peptides. Serotyping both members of the mother/child pair improves the test, i.e., among 26% of them only one member was positive. Serotype I was the most frequent (38%), which was congruent with previous genotyping results in animals and humans of the same area. This serotype was significantly more frequent among mothers who transmitted the infection to their offspring than among those who did not (53 vs. 8%, p = 0.04) and related to disease dissemination in congenitally infected children, although non-significantly. In conclusion, serotyping using the improved GRA6 peptide triad is useful to serotype T. gondii in humans and could be implemented for clinical management and epidemiological studies, to provide information on the parasite type in specific areas.