Cruzipain induces both mucosal and systemic protection against Trypanosoma cruzi in mice - PubMed (original) (raw)
Cruzipain induces both mucosal and systemic protection against Trypanosoma cruzi in mice
Anita R Schnapp et al. Infect Immun. 2002 Sep.
Abstract
Cruzipain, the major cysteinyl proteinase of Trypanosoma cruzi, is expressed by all developmental forms and strains of the parasite and stimulates potent humoral and cellular immune responses during infection in both humans and mice. This information suggested that cruzipain could be used to develop an effective T. cruzi vaccine. To study whether cruzipain-specific T cells could inhibit T. cruzi intracellular replication, we generated cruzipain-reactive CD4(+) Th1 cell lines. These T cells produced large amounts of gamma interferon when cocultured with infected macrophages, resulting in NO production and decreased intracellular parasite replication. To study the protective effects in vivo of cruzipain-specific Th1 responses against systemic T. cruzi challenges, we immunized mice with recombinant cruzipain plus interleukin 12 (IL-12) and a neutralizing anti-IL-4 MAb. These immunized mice developed potent cruzipain-specific memory Th1 cell responses and were significantly protected against normally lethal systemic T. cruzi challenges. Although cruzipain-specific Th1 responses were associated with T. cruzi protective immunity in vitro and in vivo, adoptive transfer of cruzipain-specific Th1 cells alone did not protect BALB/c histocompatible mice, indicating that additional immune mechanisms are important for cruzipain-specific immunity. To study whether cruzipain could induce mucosal immune responses relevant for vaccine development, we prepared recombinant attenuated Salmonella enterica serovar Typhimurium vaccines expressing cruzipain. BALB/c mice immunized with salmonella expressing cruzipain were significantly protected against T. cruzi mucosal infection. Overall, these data indicate that cruzipain is an important T. cruzi vaccine candidate and that protective T. cruzi vaccines will need to induce more than CD4(+) Th1 cells alone.
Figures
FIG. 1.
Purification of recombinant cruzipain. Equivalent amounts of the phage10 control protein (lanes 1 and 4) and cruzipain (lanes 2 and 5) purified from bacterial lysates were separated on 10% polyacrylamide gels next to prestained molecular mass standards (lanes 3 and 6). Lanes 1 to 3 show Coomassie staining of separated proteins. Lanes 4 to 6 show proteins blotted to nitrocellulose and probed with the 212BH6 MAb specific for cruzipain. Molecular masses in kilodaltons are indicated at the right.
FIG. 2.
Cruzipain-specific, CD4+ Th1 cells induce NO and protect against intracellular T. cruzi replication in murine macrophages. PEM were cultured in tissue culture slide chambers alone or infected with T. cruzi trypomastigotes. Cruzipain-specific T cells, ovalbumin-specific control T cells, or no T cells were added, and the cultures were incubated for 48 h prior to the supernatants being harvested for measurements of IFN-γ (a) and NO (b) production. The percentage of _T. cruzi_-infected macrophages and the average number of AMA per infected cell were counted microscopically (c). The effects of added T cells are expressed as the percent inhibition of T. cruzi infection compared with macrophages in the absence of T cells. Shown are the means ± standard errors from duplicate PEM cultures. Similar results were seen in multiple experiments.
FIG. 3.
IL-12 administered by the same route as antigen induces optimal Th1-polarized responses. Two groups of mice were immunized three times with parasite lysate. With the first two immunizations, IL-12 was given the day before, the day of, and the day after antigen administration, and IL-4-neutralizing 11B11 was given the day before and 2 days after antigen administration. The third immunization included antigen alone. Antigen was given subcutaneously (s.c.), and 11B11 was given intraperitoneally (i.p.). Group A was given IL-12 subcutaneously, and group B was given IL-12 intraperitoneally. Three days after the last immunization, LNC were harvested and stimulated in vitro with or without parasite lysate for 3 days. The supernatants were harvested and assayed for IFN-γ and IL-4 production by ELISA. Shown are the means ± standard errors. The background results from cultures incubated without parasite lysate were subtracted from the results obtained in matching cultures incubated with parasite lysate.
FIG. 4.
Immunization with cruzipain induces potent Th1 memory responses. Draining LNC were harvested from mice after immunization with phage10 control protein or cruzipain plus IL-12 and the IL-4 neutralizing MAb 11B11. CD4+ T cells were purified and cultured in vitro in the presence or absence of cruzipain for 3 days. (a) Proliferation was measured by [3H]thymidine incorporation. The results are shown as stimulation indices. (b) IFN-γ production was measured by ELISA using the supernatants collected after 3 days of stimulation. The results are shown after background IFN-γ levels present in medium-rested culture supernatants were subtracted. Antigen-specific responses are expected after immunization with phage10 protein because the recombinant cruzipain used for in vitro stimulation contains the phage10 protein as an N-terminal fusion partner. The error bars indicate standard errors.
FIG. 5.
Mice were immunized with cruzipain plus IL-12 and 11B11 and challenged with virulent T. cruzi BFT 1 month later. Three and 14 days postinfection, draining LNC were harvested and CD4+ T cells were purified. The CD4+ T cells were cultured in vitro for 3 days with medium, cruzipain, or whole T. cruzi lysate. (a) Proliferative responses to cruzipain were measured by [3H]thymidine incorporation and are shown as stimulation indices (SI). IFN-γ responses to cruzipain (b) and parasite lysate (c) were measured by ELISA. The results are shown after background levels of IFN-γ present in supernatants from cultures incubated with medium alone were subtracted. The error bars indicate standard errors. DPI, days postinfection.
FIG. 5.
Mice were immunized with cruzipain plus IL-12 and 11B11 and challenged with virulent T. cruzi BFT 1 month later. Three and 14 days postinfection, draining LNC were harvested and CD4+ T cells were purified. The CD4+ T cells were cultured in vitro for 3 days with medium, cruzipain, or whole T. cruzi lysate. (a) Proliferative responses to cruzipain were measured by [3H]thymidine incorporation and are shown as stimulation indices (SI). IFN-γ responses to cruzipain (b) and parasite lysate (c) were measured by ELISA. The results are shown after background levels of IFN-γ present in supernatants from cultures incubated with medium alone were subtracted. The error bars indicate standard errors. DPI, days postinfection.
FIG. 5.
Mice were immunized with cruzipain plus IL-12 and 11B11 and challenged with virulent T. cruzi BFT 1 month later. Three and 14 days postinfection, draining LNC were harvested and CD4+ T cells were purified. The CD4+ T cells were cultured in vitro for 3 days with medium, cruzipain, or whole T. cruzi lysate. (a) Proliferative responses to cruzipain were measured by [3H]thymidine incorporation and are shown as stimulation indices (SI). IFN-γ responses to cruzipain (b) and parasite lysate (c) were measured by ELISA. The results are shown after background levels of IFN-γ present in supernatants from cultures incubated with medium alone were subtracted. The error bars indicate standard errors. DPI, days postinfection.
FIG. 6.
Immunization with cruzipain and Th1-biasing reagents induces protective immunity against T. cruzi challenge. Mice were immunized with cruzipain plus IL-12 and the IL-4-neutralizing 11B11 MAb as described in the text. One month after the last immunization, the mice were challenged with virulent T. cruzi BFT. (a) Parasitemias measured by microscopic examination of blood samples postchallenge. For the unimmunized, phage10, and cruzipain groups, n = 5, 9, and 8, respectively. (b) Survival data postchallenge in a second immunization experiment with a more virulent T. cruzi challenge (n = 5/group). Statistically significant differences are denoted by asterisks: P < 0.05 comparing the cruzipain-immunized group with the unimmunized control group, as determined by the Mann-Whitney U test (a) or by the two-tailed Fisher's exact test (b). There were no significant differences detected in comparisons between the control groups. The error bars indicate standard errors.
FIG. 7.
A recombinant salmonella vaccine expressing cruzipain induces T. cruzi mucosal protection. Mice were vaccinated four times intranasally with recombinant salmonella expressing cruzipain (rCPSalmonella) or control salmonella transformed with the vector alone (NCSalmonella); 2 × 106 CFU of salmonella were given for primary vaccination, and 2 × 107 CFU of salmonella were given for three booster vaccinations. The second vaccination was given 4 weeks after the first dose, and the three booster vaccinations were given at 2-week intervals. One month after the last vaccination, the mice were challenged orally with T. cruzi IMT. Fourteen days after challenge, gastric tissues (where initial T. cruzi mucosal invasion occurs) were studied for levels of T. cruzi DNA by real-time PCR (a), and draining gastric LNC were examined for viable T. cruzi parasites by a limiting-dilution quantitative-culture technique (b). ∗, P < 0.05 comparing the mice given salmonella expressing cruzipain with control mice by Student's t test.
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