Immune responses against excreted/secreted antigens of Toxoplasma gondii tachyzoites in the murine model

In the present study, excretory secretory antigens (ESA) of Toxoplasma gondii were evaluated in immunization of 8-10 week inbred female Balb/c mice. Tachyzoites of the parasite were cultured in cell-free incubation medium (RPMI-1640), and then supernatant of the medium was loaded on an ion-exchange chromatography column. Two fractions (ESA-F(1) and ESA-F(2)) were collected from the column. For immunization of the mice, 50 were allocated into 5 groups of 10. The first, second, third, and fourth groups were immunized, twice with total-ESA, ESA-F(1), ESA-F(2) or toxoplasma lysate antigen (TLA), respectively. The fifth group was selected as a negative control group (non-immunized). The virulent RH strain of Toxoplasma gondii was used to challenge. Delayed-type hypersensitivity responses (DTHs) were measured by intra-footpad injection measuring induration at timed intervals. Lymphocyte transformation tests (LTTs) were done on lymph node cells using 3H] thymidine incorporation as an indication of reactivity. Peritoneal macrophages from sensitized mice were stimulated and nitric oxide was measured by Griess method. The ESA-F(1) and ESA-F(2) fractions were separated on poly acrylamide gel electrophoresis (PAGE) and SDS-PAGE. ESA-F(1) had 4 bands on PAGE and 14 bands on SDS-PAGE. ESA-F(2) had one band on PAGE and two bands on SDS-PGE. Sensitized mice showed DTH and lymphocyte transformation responses to total-ESA, ESA-F(1), and ESA-F(2) and peritoneal macrophages produce nitric oxide following stimulation. In challenge experiments, all non-immunized mice died within 10 days, whereas immunized mice survived for longer time periods (P<0.05). The highest survival rate was observed in mice that immunized with ESA-F(2). We suggest that these antigens especially ESA-F(2) should be of value for the development of new strategies for immunization against toxoplasmosis.