Intranasal vaccinations with the trans-sialidase antigen plus CpG Adjuvant induce mucosal immunity protective against conjunctival Trypanosoma cruzi challenges

Abstract

Trypanosoma cruzi is an intracellular protozoan parasite capable of infecting through mucosal surfaces. Our laboratory has previously elucidated the anatomical routes of infection after both conjunctival and gastric challenge in mice. We have shown that chronically infected mice develop strong immune responses capable of protecting against subsequent rechallenge with virulent parasites through gastric, conjunctival, and systemic routes of infection. We have also shown that intranasal immunizations with the unique T. cruzi trans-sialidase (TS) antigen protect against gastric and systemic T. cruzi challenge. In the current work we have investigated the ability of purified TS adjuvanted with CpG-containing oligonucleotides to induce immunity against conjunctival T. cruzi challenge. We confirm that intranasal vaccinations with TS plus CpG induce TS-specific T-cell and secretory IgA responses. TS-specific secretory IgA was detectable in the tears of vaccinated mice, the initial body fluid that contacts the parasite during infectious conjunctival exposures. We further show that intranasal vaccinations with TS plus CpG protect against conjunctival T. cruzi challenge, limiting local parasite replication at the site of mucosal invasion and systemic parasite dissemination. We also provide the first direct evidence that mucosal antibodies induced by intranasal TS vaccination can inhibit parasite invasion.

FIG. 1.

Intranasal vaccination with TS CpG induces proliferative immune responses and IFN-γ production. (a) Spleen cells (SC) from mice vaccinated with TS CpG showed significantly greater proliferation in response to recombinant TS protein than those from NC CpG-vaccinated controls (n = 3/group; P < 0.01 by Mann-Whitney U test). (b) SC from TS CpG-vaccinated mice had significantly higher numbers of IFN-γ-producing cells in response to both TS protein (black bars) and an H2-k^d-restricted CD8⁺ T-cell epitope of TS (hatched lines) than those from NC CpG vaccinated controls (n = 3/group; P < 0.01 by Mann-Whitney U test). In both panels, error bars represent the standard errors of the means. These results are representative of five experiments.

FIG. 2.

Intranasal TS CpG vaccination induces secretory IgA production. Fecal extracts (FE) and tears (a and b, respectively) were harvested from TS CpG- and NC CpG-vaccinated mice. End point titers were determined by TS-specific ELISA. Differences between TS CpG- and control-vaccinated mice were statistically significant to end point dilutions of 1:64 and 1:320 in FE and tears, respectively (n = 5/group; P < 0.04 and 0.02, respectively, by Student's t tests). These results are representative of three experiments. Error bars indicate standard deviations.

FIG. 3.

Intranasal TS CpG vaccination protects mice against a systemic T. cruzi challenge. BALB/c mice were vaccinated with either TS CpG or NC CpG and later challenged subcutaneously with 5,000 BFT. Five of the six TS CpG-vaccinated mice survived to an end point of 50 days, while none of the NC CpG-vaccinated mice survived (n = 6/group; P < 0.02 by Fisher's exact two-tailed test). These results are representative of five experiments.

FIG. 4.

Intranasal TS CpG vaccination induces immune responses and sIgA protective against conjunctival T. cruzi challenge. (a) Intranasally TS CpG- and NC CpG-vaccinated mice were challenged conjunctivally 1 month after their second vaccination and sacrificed 10 days later, and draining lymph nodes were harvested. The numbers of parasite molecular equivalents (ME) were determined by real-time PCR. Significantly lower levels of recoverable parasite DNA were found in the lymph nodes of TS CpG-vaccinated mice than in those of negative controls (n = 6 and 9/group, respectively; P < 0.04 by Mann-Whitney U test). These results are representative of three experiments. (b) Parasites were opsonized with filtered FE from TS CpG-vaccinated or control mice for 30 min. Naïve BALB/c mice were challenged conjunctivally with opsonized parasite suspensions. At 10 days after challenge, DNA was isolated from the draining lymph nodes and parasite levels assayed by real-time PCR. Challenges with parasites opsonized with FE from TS CpG-vaccinated mice resulted in significantly lower levels of infection than challenges with parasites opsonized with FE from control mice (P < 0.03 by Mann-Whitney U test). These results are from a single experiment with n = 6 per group. Error bars indicate standard deviations.