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Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi.We designed a high-density peptide microarray containing more than 175,000 overlapping 15mer peptides derived from T. cruziproteins. Peptides were synthesized in situon microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a 10-fold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ∼threefold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens.

ABSTRACTThe trans-sialidases (TSs) from Trypanosoma cruzi, the agent of Chagas disease, are virulence factors shed to the bloodstream that induce strong alterations in the immune system. Here, we report that both enzymatically active TS (aTS) and its lectinlike isoform (iTS) disturb CD4 T cell physiology, inducing downregulation of Th1 cell functionality and in vivocell expansion. By using ovalbumin-specific DO11.10 cells as tracers of clones developing the Th1 phenotype, we found that the infection induced significant amounts of gamma interferon (IFN-γ) but low levels of interleukin 2 (IL-2) and increased IL-4 production in vivo, in agreement with a mixed T helper response. The production of cytokines associated with the Th2 phenotype was prevented by passive transfer of anti-TS neutralizing antibodies. TSs also reduced the T cell receptor signaling as assayed by Zap-70 phosphorylation. TSs also reduced IL-2 and IFN-γ secretion, with a concomitant increase in IL-4 production and then an unbalancing of the CD4 T cell response toward the Th2 phenotype. This effect was prevented by using anti-IL-10 neutralizing antibodies or IL-10−/−antigen-presenting cells, supporting the subversion of this regulatory pathway. In support, TSs stimulated IL-10 secretion by antigen-presenting cells during their interaction with CD4 T cells. When polarized cells were stimulated in the presence of TSs, the secretion of IL-2 and IFN-γ was strongly downregulated in Th1 cells, while IL-2 production was upregulated in Th2 cells. Although the Th1 response is associated with host survival, it may simultaneously induce extensive damage to infected tissues. Thus, by delaying the elicitation of the Th1 response and limiting its effector properties, TSs restrain the cell response, supporting T. cruzicolonization and persistence while favoring host survival.

CD4+ T cells are the main lymphocyte target of Galectin‐8, which lowers their activation threshold and thus suggesting the lectin involvement in inflammatory diseases. Galectin (Gal) constitute a family of carbohydrate‐recognizing molecules ubiquitously expressed in mammals. In the immune system, they regulate many processes such as inflammation, adhesion, and apoptosis. Here, we report the expression in the spleen of the two same Gal‐8 splice variants described previously in the thymus. Gal‐8 was found to induce two separate biological activities on T lymphocytes: a robust naive CD4+T cell proliferation in the absence of antigen and notably, a costimulatory signal that synergized the cognate OVA peptide in DO11.10 mice transgenic for TCROVA. The antigen‐independent proliferation induced by Gal‐8 displayed increased expression of pro‐ and anti‐inflammatory cytokines, thus suggesting the polyclonal expansion of Th1 and Th2 clones. The costimulatory effect on antigen‐specific T cell activation was evidenced when the Gal and the peptide were assayed at doses suboptimal to induce T cell proliferation. By mass spectra analysis, several integrins and leukocyte surface markers, including CD45 isoforms, as well as other molecules specific to macrophages, neutrophils, and platelets, were identified as putative Gal‐8 counter‐receptors. Gal‐8 triggered pZAP70 and pERK1/2. Moreover, pretreatment with specific inhibitors of CD45 phosphatase or ERK1/2 prevented its antigen‐dependent and ‐independent T cell‐proliferative activities. This seems to be associated with the agonistic binding to CD45, which lowers the activation threshold of the TCR signaling pathway. Taken together, our findings support a distinctive role for locally produced Gal‐8 as an enhancer of otherwise borderline immune responses and also suggest that Gal‐8 might fuel the reactivity at inflammatory foci.

ABSTRACTStrong thrombocytopenia is observed during acute infection with Trypanosoma cruzi, the parasitic protozoan agent of American trypanosomiasis or Chagas' disease. The parasite sheds trans-sialidase, an enzyme able to mobilize the sialyl residues on cell surfaces, which is distributed in blood and is a virulence factor. Since the sialic acid content on the platelet surface is crucial for determining the half-life of platelets in blood, we examined the possible involvement of the parasite-derived enzyme in thrombocytopenia induction. We found that a single intravenous injection of trans-sialidase into naïve mice reduced the platelet count by 50%, a transient effect that lasted as long as the enzyme remained in the blood. CD43−/−mice were affected to a similar extent. When green fluorescent protein-expressing platelets were treated in vitro with trans-sialidase, their sialic acid content was reduced together with their life span, as determined after transfusion into naïve animals. No apparent deleterious effect on the bone marrow was observed. A central role for Kupffer cells in the clearance of trans-sialidase-altered platelets was revealed after phagocyte depletion by administration of clodronate-containing liposomes and splenectomy. Consistent with this, parasite strains known to exhibit more trans-sialidase activity induced heavier thrombocytopenia. Finally, the passive transfer of a trans-sialidase-neutralizing monoclonal antibody to infected animals prevented the clearance of transfused platelets. Results reported here strongly support the hypothesis that the trans-sialidase is the virulence factor that, after depleting the sialic acid content of platelets, induces the accelerated clearance of the platelets that leads to the thrombocytopenia observed during acute Chagas' disease.

ABSTRACTTrypanosoma cruzi, the agent of Chagas' disease, expresses trans-sialidase, a unique enzyme activity that enables the parasite to invade host cells by transferring sialyl residues from host glyconjugates to the parasite's surface acceptor molecules. The enzyme is also shed into the surrounding environment, causing apoptosis in cells from the immune system. During infections, an antibody response against the catalytic region of the trans-sialidase that is coincident with the control of the parasitemia and survival of the host is observed. This low-titer humoral response is characterized by its persistence for many years in benznidazole-treated patients. Here we analyzed the antigenic structure of the molecule by phage-displayed peptide combinatorial libraries and SPOT synthesis. Several epitopes were defined and located on the three-dimensional model of the enzyme. Unexpectedly, cross-reaction was found among several epitopes distributed in different locations displaying nonconsensus sequences. This finding was confirmed by the reactivity of three monoclonal antibodies able to recognize non-sequence-related peptides that together constitute the surface surrounding the catalytic site of the enzyme. The presence of cross-reacting epitopes within a single molecule suggests a mechanism developed to avoid a strong humoral response by displaying an undefined target to the immune system.

Proliferation of Leishmania mexicana promastigotes in synthetic medium can be blocked by the depletion of intracellular polyamine pools induced by the presence of d,l-α-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC). Here we report that DFMO-resistant cell lines growing normally at DFMO levels of 10 mM have been obtained from non-proliferating cultures after a single-step selection in the presence of high concentrations of the drug. The DFMO-resistant promastigotes underwent a morphological transformation into an ‘amastigote-like’ form after incubation for several hours at gradually increasing temperatures up to 35 °C. The uptake of DFMO was not significantly altered in the drug-resistant cell lines but in both cases (promastigote and ‘amastigote-like’ forms) the ODC specific activity was increased approx. 15-fold over the normal enzymic levels found in the wild-type Leishmania. The enzyme affinities for its substrate and for DFMO gave very similar values in the drug-resistant promastigotes and the wild-type parasites. In contrast, ODC from the ‘amastigote-like’Leishmania showed a higher affinity for ornithine and a decreased capacity for the binding of DFMO. An 80-fold amplification of the ODC gene and a corresponding increase in its transcripts have been detected in both DFMO-resistant Leishmania cell lines. The drug-resistant phenotypes with their characteristic morphologies, the increased levels of ODC activity and the amplification of the ODC gene have been stable for at least 6 months in the absence of selective pressure.