Type 1 immunity provides optimal protection against both mucosal and systemic Trypanosoma cruzi challenges

Abstract

Chagas' disease results from infection with Trypanosoma cruzi, a protozoan parasite that establishes systemic intracellular infection after mucosal invasion. We hypothesized that ideal vaccines for mucosally invasive, intracellular pathogens like T. cruzi should induce mucosal type 2 immunity for optimal induction of protective secretory immunoglobulin A (IgA) and systemic type 1 immunity protective against intracellular replication. However, differential mucosal and systemic immune memory could be difficult to induce because of reciprocal inhibitory actions between type 1 and type 2 responses. To test our hypotheses, we investigated the protective effects of type 1 and type 2 biased vaccines against mucosal and systemic T. cruzi challenges. Intranasal vaccinations were given with recombinant interleukin-12 (IL-12)- and IL-4-neutralizing antibody (Ab) for type 1 immune bias, or recombinant IL-4 and gamma interferon-neutralizing Ab for type 2 immune bias. Cytokine RNA and protein studies confirmed that highly polarized memory immune responses were induced by our vaccination protocols. Survival after virulent subcutaneous T. cruzi challenge was used to assess systemic protection. Mucosal protection was assessed by measuring the relative inhibition of parasite replication in mucosal tissues early after oral T. cruzi challenge, using both PCR and quantitative culture techniques. As expected, only type 1 responses protected against systemic challenges (P < 0.01). However, contrary to our original hypothesis, type 1 responses optimally protected against mucosal challenges as well (P < 0.05). Type 1 and type 2 biased vaccines induced similar secretory IgA responses. We conclude that future vaccines for T. cruzi and possibly other mucosally invasive, intracellular pathogens should induce both mucosal and systemic type 1 immunity.

FIG. 1.

Intranasal vaccinations with no bias, type 1 bias, and type 2 bias induce similar T. cruzi-specific lymphoproliferation. Mice were immunized as described in Table 1, and spleen cells were harvested 3 days following the final intranasal immunization. (A) Total spleen cells (2 × 10⁵/200 μl) were stimulated with 2 or 20 μg of T. cruzi lysate/ml for 72 h prior to measurement of [³H]thymidine incorporation. (B) A total of 5 × 10⁴ CD4⁺ purified spleen cells/well and 10⁶ irradiated syngeneic total spleen cells/well were stimulated in 96-well plates with 2 or 20 μg of T. cruzi lysate/ml for 72 h prior to measurement of [³H]thymidine incorporation. Shown are the means and standard errors from a single experiment with pooled cells from two to three mice/group. These results are representative of at least five similar experiments.

FIG. 2.

RNase protection assays demonstrate that the type 1 and type 2 biased intranasal vaccination protocols induced highly polarized immune responses. Total spleen cells (4 × 10⁶ cells/ml) were harvested and rested in medium alone, stimulated with T. cruzi lysate, or stimulated with ConA, using methods similar to those described in the legend to Fig. 1. Total RNA samples were harvested from parallel cultures 24 and 48 h later using RNeasy Kits (Qiagen). These RNA samples were used in RiboQuant RNase protection assays (Pharmingen) to assess the levels of cytokine mRNA expressed. The autoradiogram shown is representative of multiple similar experiments. A progressive increase in IFN-γ mRNA expression induced by T. cruzi lysate in vitro was the most prominent finding with spleen cells from type 1 biased mice. On the other hand, parasite-specific increases in IL-4, IL-5, IL-9, and IL-13 mRNA were detected with spleen cells from type 2 biased mice, especially after 48 h of in vitro stimulation.

FIG. 3.

Cytokine secretion studies confirm that type 1 and type 2 biased immunization protocols induce highly polarized T. cruzi-specific immunity. BALB/c mice were immunized as described in Table 1. Three days after the final intranasal antigen challenge, mice were sacrificed and spleen cell suspensions were prepared. (A) Total spleen cells (2 × 10⁵ cells/200 μl) were stimulated with T. cruzi lysate, and 72 h later the culture supernatants were harvested and assayed for secreted IFN-γ and IL-4 levels by ELISA. (B) Similar studies were done with purified CD4⁺ T cells (5 × 10⁴ cells/200 μl) cultured with irradiated syngeneic total spleen cells (10⁶ cells/200 μl) pulsed with T. cruzi lysate. Both sets of data had background levels of cytokines spontaneously produced in medium-rested cultures subtracted from the values presented. Shown are the means and standard errors from a single experiment with pooled cells derived from two to three mice/group. These results are representative of at least five similar experiments. Similar results were seen in lymphocytes harvested from Peyer's patches and from mesenteric lymph nodes (data not shown).

FIG. 4.

Nonbiased, type 1 biased, and type 2 biased intranasal vaccinations induced similar levels of T. cruzi-specific secretory IgA responses. Fecal extracts collected from mice after vaccination were studied in an IgA-specific ELISA with T. cruzi lysate. Shown are mean ELISA ODs (± the standard error) for serial dilutions in each group. Similar results were seen in multiple experiments. Calculated endpoint titers (dilution with an OD double the OD of negative controls) were between 1/30 and 1/100 for all three groups (nonbiased, type 1 biased, and type 2 biased) in all experiments. Intranasal administration of cholera toxin alone did not increase ELISA reactivity of fecal extracts with T. cruzi lysate (data not shown).

FIG. 5.

SQ-PCR assessment of T. cruzi replication in mucosal tissues. DNA samples were extracted from mucosal tissues using QIAamp tissue kits and used as templates in PCRs with primers specific for the C-terminal 418-bp fragment of the major T. cruzi cysteinyl proteinase, cruzipain. A constant amount of each DNA sample was amplified with 10-fold dilutions of a positive control DNA fragment. The positive control contains the same 5′ and 3′ ends as the cruzipain C terminus but is smaller by 25 bp than the wild-type sequence, making it distinguishable in ethidium bromide-stained 2% TBE agarose gels. The point of equivalent band intensity for both positive control and native PCR products can be used to assess the relative quantities of T. cruzi DNA in unknown samples. Shown are four sets of five reactions with 10-fold dilutions of the positive control, each set with known samples containing 10-fold-decreasing amounts of T. cruzi DNA. Reactions in lanes 2 to 6, 7 to 11, 13 to 17, and 18 to 22 contained 500, 50, 5, and 0 parasite equivalents of T. cruzi DNA, respectively. Lanes 1 and 12 were loaded with a 123-bp ladder molecular mass standard.

FIG. 6.

Type 1 responses provide optimal protection against T. cruzi mucosal infection. BALB/c mice were immunized with nonbiased, type 1 biased, and type 2 biased vaccination protocols as described in Table 1. One month after the last immunization, mice were challenged orally with 10,000 T. cruzi IMT. Ten days after oral IMT challenge, gastric DNA samples were isolated and studied by using T. cruzi-specific SQ-PCR as described in the legend to Fig. 5. Nonbiased and type 2 biased mice had lower levels of recoverable T. cruzi DNA compared with unvaccinated animals (P < 0.05 by the Mann-Whitney U test). However, type 1 biased mice were optimally protected against mucosal T. cruzi challenge, with 3 log-greater reductions in recovered parasite DNA compared with nonbiased and type 1 biased mice (P < 0.05 by the Mann-Whitney U test). Similar results were detected in multiple experiments.

FIG. 7.

Type 1 biased immunization inhibits recovery of viable T. cruzi parasites from mucosal draining lymph nodes after oral IMT challenge. Within 1 week after delivery of oral IMT, an enlarged lymph node is visible within the lesser curvature of the stomach of challenged mice. This enlarged node represents the initial lymphatic drainage site proximal to the point of mucosal invasion. Single-cell preparations of lymphocytes from these gastric lymph nodes can be harvested and serially diluted in LDNT+ medium ideal for outgrowth of T. cruzi epimastigotes. After incubation at 26°C for 1 to 2 months, the minimal number of lymphocytes associated with detectable epimastigote outgrowth is directly proportional to the levels of replicating parasites present in the original gastric lymph node tissues. The minimal number of parasites per million lymphocytes present within the original lymph nodes is calculated by dividing 1 million by the minimal number of lymphocytes associated with parasite outgrowth. Both nonbiased and type 1 biased mice were significantly protected against T. cruzi replication in mucosal draining lymph nodes compared with unvaccinated and type 2 biased mice (P < 0.05 by the Mann-Whitney U test). Similar results were detected in multiple experiments.