Toxoplasma gondii-Derived Synthetic Peptides Containing B- and T-Cell Epitopes from GRA2 Protein Are Able to Enhance Mice Survival in a Model of Experimental Toxoplasmosis

Abstract

Toxoplasmosis is a zoonosis distributed all over the world, which the etiologic agent is an intracellular protozoan parasite, Toxoplasma gondii. This disease may cause abortions and severe diseases in many warm-blood hosts, including humans, particularly the immunocompromised patients. The parasite specialized secretory organelles, as micronemes, rhoptries and dense granules, are critical for the successful parasitism. The dense granule protein 2 (GRA2) is a parasite immunogenic protein secreted during infections and previous studies have been shown that this parasite component is crucial for the formation of intravacuolar membranous nanotubular network (MNN), as well as for secretion into the vacuole and spatial organization of the parasites within the vacuole. In the present study, we produced a monoclonal antibody to GRA2 (C3C5 mAb, isotype IgG2b), mapped the immunodominant epitope of the protein by phage display and built GRA2 synthetic epitopes to evaluate their ability to protect mice in a model of experimental infection. Our results showed that synthetic peptides for B- and T-cell epitopes are able to improve survival of immunized animals. In contrast with non-immunized animals, the immunized mice with both B- and T-cell epitopes had a better balance of cytokines and demonstrated higher levels of IL-10, IL-4 and IL-17 production, though similar levels of TNF-α and IL-6 were observed. The immunization with both B- and T-cell epitopes resulted in survival rate higher than 85% of the challenged mice. Overall, these results demonstrate that immunization with synthetic epitopes for both B- and T-cells from GRA2 protein can be more effective to protect against infection by T. gondii.

Keywords: B- and T-cell epitopes; GRA2; Toxoplasma gondii; monoclonal antibody.

Figure 1

Selection and immunochemical characterization of monoclonal antibody C3C5 specific for GRA2 protein of Toxoplasma gondii. (A) Isotyping of C3C5 mAb by using an immunoenzymatic assay in culture supernatant. (B) 1D immunoblot of T. gondii RH strain tachyzoites solubilized in SDS sample buffer and resolved in 12% SDS-PAGE and probed with no infected mouse serum sample (Lane 1), T. gondii infected mouse serum sample (Lane 2), C3C5 mAb (Lane 3), and anti-p30 mAb E9 (Lane 4). (C) 2D immunoblot of T. gondii RH strain tachyzoites solubilized in isoelectric focalization solution and resolved through pH 3–10 and then in 12% SDS-PAGE. After electrotransference to nitrocellulose membranes, antigens were probed with C3C5 mAb. (D) Indirect fluorescent antibody test (IFAT) of T. gondii RH strain probed with mice serum no infected (−), infected with T. gondii (+) and monoclonal antibody C3C5. Slides containing parasite were permeabilized (+) or not (−) with Triton X-100 0.01% for 10 min. (E) Proliferation inhibition of T. gondii infection by C3C5 mAb in HeLa. T. gondii 2F1 strain tachyzoites were pretreated for 60 min at 37°C, 5% CO₂ with increased concentration (1.56–100 μg/mL) of C3C5 mAb and irrelevant mouse IgG. The proliferation assay was development from β–gal activity measured at absorbance of 550 nm using a kinetic plate reader (Molecular Devices).

Figure 2

Epitope mapping of GRA2 by Phage Display and peptides synthesis. (A) Peptide sequences of 24 phage clones and their consensus sequences, according to the original sequence determined for the C-terminal region of the GRA2 protein. (B) Immunodominant region especially for B cell (http://iedb.org/). (C) Based on the results of prediction for B cells and T cells into GRA2 structure was designed to be synthesized two peptides, Tx1 and Tx2, respectively.

Figure 3

Structural modeling of dense granule 2 (GRA2) of Toxoplasma gondii. In silico analysis of three-dimensional structure of GRA2 protein were performed to search for possible intrinsically unstructured protein (IUP) domains, evidencing predicted epitopes for B (highlighted in yellow) and T (highlighted in red).

Figure 4

Survival curves of C57BL/6 mice after challenge with Toxoplasma gondii. Five groups of mice (10 animals per group) were immunized with soluble tachyzoite antigen (STAg); bovine serum albumin (BSA); synthetic peptide Tx1 (Tx1); synthetic peptide Tx2 (Tx2) and both synthetic peptides (Tx1+Tx2). All curves are compared to the group immunized only with BSA. (A) Survival of the BSA group (28.6%) and survival group STAg (71.4%). (B) Survival of the BSA group (28.6%) and survival group Tx1 (42.8 %). (C) Survival of the BSA group (28.6%) and survival group Tx2 (71.4 %). (D) Survival of the BSA group (28.6%) and survival group Tx1+Tx2 (85.7%). This group showed significant difference in survival. ^*P < 0.05.

Figure 5

Levels of IgG, IgG1, and IgG2a anti-STAg (A) or anti-Tx1+Tx2 (B) determined by ELISA in serum samples from C57BL/6 mice immunized with STAg, BSA or Tx1+Tx2. It was observed that animals immunized with STAg recognized only STAg and animals immunized with Tx1+Tx2 only recognized Tx1+Tx2.

Figure 6

Cytokine production by mice immunized with STAg, BSA, or Tx1+Tx2 after challenge with T. gondii. (A–D) Pro-inflammatory cytokine profiles. (E,F) Anti-inflammatory cytokine profile. (G) IL-17 production. The group immunized with Tx1+Tx2 presented a better balance between pro- and anti-inflammatory cytokines.