Antigen and antibody levels of TgPrx1 in body fluids of infected mice.

<p>(A) C57BL/6 mice (n = 4) were intraperitoneally infected with 10³ <i>T</i>. <i>gondii</i> PLK or RH tachyzoites. TgPrx1 antigen was measured in ascitic fluid collected from experimentally infected mice at 0, 3, and 6 days postinfections (dpi). Each value represents the mean ± standard deviation of quadruplicate samples. nd, not detected. *, statistically significant differences were observed between the two groups with the Student’s <i>t</i> test (<i>P</i> < 0.05). <b>(B)</b> Production of IgG antibodies against TgPrx1 in chronically infected mice. C57BL/6 mice (n = 5) were intraperitoneally infected with 10³ <i>T</i>. <i>gondii</i> PLK tachyzoites. Serum samples were collected from the mice 4 weeks after infection and tested with indirect ELISAs, using recombinant TgPrx1-GST and TgGRA7 antigens. The mean optical density (OD) was determined at a wavelength of 415 nm. Each bar represents the mean ± standard deviation. Sera of uninfected mice (n = 4) were used as the negative control. *, statistically significant differences were observed between the uninfected and infected mice with the Student’s <i>t</i> test (<i>P</i> < 0.05).</p

Survival of mice and parasite numbers in brains of surviving mice.

<p>(A) Six mice per group were immunized with TgPrx1-GST, GST, or PBS, and then challenged with 1 × 10³ tachyzoites of the <i>T</i>. <i>gondii</i> PLK strain. The survival rates (surviving mice/total mice) are calculated from three pooled independent experiments: PBS, 5/18 (27.8%); TgPrx1, 12/18 (66.7%); GST, 7/18 (38.9%). *, statistically significant differences in the survival rates at 30 days postinfection (dpi) were observed between the PBS-injected group and the recombinant-protein-immunized groups with a χ² test (<i>P</i> < 0.05). (B) Parasite numbers in the brains of the surviving mice at 30 dpi. Results are from three pooled independent experiments (PBS, n = 5; TgPrx1, n = 12; GST, n = 7). The results were analyzed with one-way ANOVA plus a Tukey–Kramer <i>post hoc</i> analysis, but there were no significant differences.</p

Immunization with <i>Toxoplasma gondii</i> peroxiredoxin 1 induces protective immunity against toxoplasmosis in mice

<div><p>To develop a vaccine against <i>Toxoplasma gondii</i>, a vaccine antigen with immune-stimulating activity is required. In the present study, we investigated the immunogenicity and prophylactic potential of <i>T</i>. <i>gondii</i> peroxiredoxin 1 (TgPrx1). The TgPrx1 was detected in the ascitic fluid of mice 6 days postinfection, while specific antibody levels were low in the sera of chronically infected mice. Treatment of murine peritoneal macrophages with recombinant TgPrx1 triggered IL-12p40 and IL-6 production, but not IL-10 production. In response to TgPrx1, activation of NF-kB and IL-6 production were confirmed in mouse macrophage cell line (RAW 264.7). These results suggest the immune-stimulating potentials of TgPrx1. Immunization of mice with recombinant TgPrx1 stimulated specific antibody production (IgG1 and IgG2c). Moreover, spleen cell proliferation and interferon-gamma production significantly increased in the TgPrx1- sensitized cells from mice immunized with the same antigen. Immunization with TgPrx1 also increased mouse survival and decreased cerebral parasite burden against lethal <i>T</i>. <i>gondii</i> infection. Thus, our results suggest that TgPrx1 efficiently induces humoral and cellular immune responses and is useful as a new vaccine antigen against toxoplasmosis.</p></div

Effects of recombinant TgPrx1 on RAW cell lines.

<p>NF-kB/SEAP cells (A) and RAW 264.7 cells (B) were treated with 10 ng/mL LPS and recombinant TgPrx1-GST or GST protein for 48 h in the presence or absence of 20 μg/mL polymixin B to measure the SEAP and IL-6, respectively. Each value represents the mean ± standard deviation of quadruple samples. The different letters above the bars in the graphs indicate statistically significant differences among the test groups and the mock group (one-way ANOVA plus Tukey–Kramer <i>post hoc</i> analysis, <i>P</i> < 0.05).</p

Expression of recombinant proteins and detection of TgPrx1 using specific antibodies.

<p>(A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of recombinant proteins, with Coomassie Blue staining. Lanes: M, molecular mass marker; lane 1, TgPrx1–GST; lane 2, GST. (B) Western blot using lysate of <i>T</i>. <i>gondii</i> tachyzoites (RH and PLK strains) using mouse and rabbit anti-TgPrx1 sera.</p

Production of IL-6, IL-12p40 and IL-10 by murine peritoneal macrophages.

<p>Murine peritoneal macrophages were treated with 1 ng/mL LPS and recombinant TgPrx1-GST or GST protein for 20 h in the presence or absence of 1 μg/mL polymixin B. The levels of IL-6 (A), IL-12p40 (B) and IL-10 (C) value represents the mean ± standard deviation of quadruple samples. The results are representative of three repeated experiments with similar results. The different letters above the bars in the graphs indicate statistically significant differences among the test groups and the mock group (one-way ANOVA plus Tukey–Kramer <i>post hoc</i> analysis, <i>P</i> < 0.05).</p