Immune Stimulation of RAP domain binding protein (rTgRA15) from Toxoplasma gondii

Abstract

Toxoplasmosis, a parasitic disease in human and animals, is caused by Toxoplasma gondii. Our previous study has led to the discovery of a novel RAP domain binding protein antigen (TgRA15), an apparent in-vivo induced antigen recognised by antibodies in acutely infected individuals. This study is aimed to evaluate the humoral response and cytokine release elicited by recombinant TgRA15 protein in C57BL/6 mice, demonstrating its potential as a candidate vaccine for Toxoplasma gondii infection. In this study, the recombinant TgRA15 protein was expressed in Escherichia coli, purified and refolded into soluble form. C57BL/6 mice were immunised intradermally with the antigen and CASAC (Combined Adjuvant for Synergistic Activation of Cellular immunity). Antigen-specific humoral and cell-mediated responses were evaluated using Western blot and ELISA. The total IgG, IgG₁ and IgG_2a antibodies specific to the antigen were significantly increased in treatment group compare to control group. A higher level of interferon gamma (IFN-γ) secretion was demonstrated in the mice group receiving booster doses of rTgRA15 protein, suggesting a potential Th1-mediated response. In conclusion, the rTgRA15 protein has the potential to generate specific antibody response and elicit cellular response, thus potentially serve as a vaccine candidate against T. gondii infection.

Keywords: TgRA; Toxoplasmosis; antibody; immunity; interferon gamma; mice; vaccine.

Figure 1.

Immunisation regimen for rTgRA15 protein in C57BL/6 mouse model. A total of five mice (n = 5) were used in each control and treatment groups, which were injected with PBS+ CASAC and rTgRA15+ CASAC, respectively. The treatment mice were initially primed with 400 µg protein, followed by five booster doses with 200 µg protein in 2-weeks interval at day 14, 28, 42, 56 and 70. At day 66 and 80, each mouse sera and PBMC was harvested for respective evaluation of IgG antibody response and IFN-γ secretion.

Figure 2.

Detection of histidine-tagged TgRA15 protein in Western blot analysis. An intense band was observed at 29 kDa in E1 (4.5 µg) and E2 (2 µg) fraction eluted from electrophoresis. No significant band was observed in the negative control containing protein without having histidine fusion tag. M indicated the PageRuler^TM prestained protein ladder as reference. An intense band was observed at ~ 29 kDa as shown by the black arrow.

Figure 3.

The antigen-specific IgG antibody response in C57BL/6 mouse model. The rTgRA15 treatment group has shown a significant higher production of IgG₁ (a), IgG_2a (b) and total IgG (c) antibody level as compared to PBS control group. The respective IgG, IgG₁ and IgG_2a monoclonal antibody was used as the positive control in the sandwich ELISA analysis. Data are presented as mean ± standard deviation and significance level is determined at ****p < 0.0001 (n = 5).

Figure 4.

Interferon gamma (IFN-γ) secretion against rTgRA15 antigen. The treatment group had significantly higher level of IFN-γ production than PBS control group upon 4^th (a) and 5^th (b) boost of rTgRA15 antigen, respectively. Concanavalin A (1 µg/ml) was used as the positive control, showing no significance difference compare to rTgRA15 treatment group. Level of mouse IFN-γ in each sample was determined from the standard curve (c) of absorbance (405nm) versus concentration of serially-diluted recombinant interferon-gamma (pg/ml). Data are presented as mean ± standard deviation and significance level is determined at *p < 0.05 and **p < 0.01 (n = 5).