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Chagas disease (ChD), caused by the protozoan parasite Trypanosoma cruzi, affects millions of people worldwide. Chemotherapy is restricted to two drugs, which are partially effective and may cause severe side effects, leading to cessation of treatment in a significant number of patients. Currently, there are no biomarkers to assess therapeutic efficacy of these drugs in the chronic stage. Moreover, no preventive or therapeutic vaccines are available. In this chapter, we describe the purification of Trypanosoma cruzi trypomastigote-derived glycosylphosphatidylinositol (GPI)-anchored mucins (tGPI-mucins) for their use as antigens for the reliable primary or confirmatory diagnosis and as prognostic biomarkers for early assessment of cure following ChD chemotherapy. We also describe, as an example, the synthesis of a potential tGPI-mucin-derived α-Gal-terminating glycan and its coupling to a carrier protein for use as diagnostic and prognostic biomarker in ChD.

Background: Protozoan parasites from the genus Leishmania cause broad clinical manifestations known as leishmaniases, which affect millions of people worldwide. Cutaneous leishmaniasis (CL), caused by L. major, is one the most common forms of the disease in the Old World. There is no preventive or therapeutic human vaccine available for L. major CL, and existing drug treatments are expensive, have toxic side effects, and resistant parasite strains have been reported. Hence, further therapeutic interventions against the disease are necessary. Terminal, non-reducing, and linear α-galactopyranosyl (α-Gal) epitopes are abundantly found on the plasma membrane glycolipids of L. major known as glycoinositolphospholipids. The absence of these α-Gal epitopes in human cells makes these glycans highly immunogenic and thus potential targets for vaccine development against CL., Methodology/principal Findings: Here, we evaluated three neoglycoproteins (NGPs), containing synthetic α-Gal epitopes covalently attached to bovine serum albumin (BSA), as vaccine candidates against L. major, using α1,3-galactosyltransferase-knockout (α1,3GalT-KO) mice. These transgenic mice, similarly to humans, do not express nonreducing, linear α-Gal epitopes in their cells and are, therefore, capable of producing high levels of anti-α-Gal antibodies. We observed that Galα(1,6)Galβ-BSA (NGP5B), but not Galα(1,4)Galβ-BSA (NGP12B) or Galα(1,3)Galα-BSA (NGP17B), was able to significantly reduce the size of footpad lesions by 96% in comparison to control groups. Furthermore, we observed a robust humoral and cellular immune response with production of high levels of protective lytic anti-α-Gal antibodies and induction of Th1 cytokines., Conclusions/significance: We propose that NGP5B is an attractive candidate for the study of potential synthetic α-Gal-neoglycoprotein-based vaccines against L. major infection.

The protozoan parasite, Trypanosoma cruzi, the etiologic agent of Chagas disease (ChD), has a cell surface covered by immunogenic glycoconjugates. One of the immunodominant glycotopes, the trisaccharide Galα(1,3)Galβ(1,4)GlcNAcα, is expressed on glycosylphosphatidylinositol-anchored mucins of the infective trypomastigote stage of T. cruzi and triggers high levels of protective anti-α-Gal antibodies (Abs) in infected individuals. Here, we have efficiently synthesized the mercaptopropyl glycoside of that glycotope and conjugated it to maleimide-derivatized bovine serum albumin (BSA). Chemiluminescent-enzyme-linked immunosorbent assay revealed that Galα(1,3)Galβ(1,4)GlcNAcα-BSA is recognized by purified anti-α-Gal Abs from chronic ChD patients ∼230-fold more strongly than by anti-α-Gal Abs from sera of healthy individuals (NHS anti-α-Gal). Similarly, the pooled sera of chronic Chagas disease patients (ChHSP) recognized Galα(1,3)Galβ(1,4)GlcNAcα ∼20-fold more strongly than pooled NHS. In contrast, the underlying disaccharide Galβ(1,4)GlcNAcα and the monosaccharide GlcNAcα or GlcNAcβ conjugated to BSA are poorly or not recognized by purified anti-α-Gal Abs or sera from Chagasic patients or healthy individuals. Our results highlight the importance of the terminal Galα moiety for recognition by Ch anti-α-Gal Abs and the lack of Abs against nonself Galβ(1,4)GlcNAcα and GlcNAcα glycotopes. The substantial difference in binding of Ch vs. NHS anti-α-Gal Abs to Galα(1,3)Galβ(1,4)GlcNAcα-BSA suggests that this neoglycoprotein (NGP) might be suitable for experimental vaccination. To this end, the Galα(1,3)Galβ(1,4)GlcNAcα-BSA NGP was then used to immunize α1,3-galactosyltransferase-knockout mice, which produced antibody titers 40-fold higher as compared with pre-immunization titers. Taken together, our results indicate that the synthetic Galα(1,3)Galβ(1,4)GlcNAcα glycotope coupled to a carrier protein could be a potential diagnostic and vaccine candidate for ChD., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chagas disease (ChD), caused by the hemoflagellate parasite Trypanosoma cruzi, affects six to seven million people in Latin America. Lately, it has become an emerging public health concern in nonendemic regions such as North America and Europe. There is no prophylactic or therapeutic vaccine as yet, and current chemotherapy is rather toxic and has limited efficacy in the chronic phase of the disease. The parasite surface is heavily coated by glycoproteins such as glycosylphosphatidylinositol (GPI)-anchored mucins (tGPI-mucins), which display highly immunogenic terminal nonreducing α-galactopyranosyl (α-Gal)-containing glycotopes that are entirely absent in humans. The immunodominant tGPI-mucin α-Gal glycotope, the trisaccharide Galα1,3Galβ1,4GlcNAc (Galα3LN), elicits high levels of protective T. cruzi-specific anti-α-Gal antibodies in ChD patients in both the acute and chronic phases. Although glycoconjugates are the major parasite glycocalyx antigens, they remain completely unexplored as potential ChD vaccine candidates. Here we investigate the efficacy of the T. cruzi immunodominant glycotope Galα3LN, covalently linked to a carrier protein (human serum albumin (HSA)), as a prophylactic vaccine candidate in the acute model of ChD, using the α1,3-galactosyltransferase-knockout (α1,3GalT-KO) mouse, which mimics the human immunoresponse to α-Gal glycotopes. Animals vaccinated with Galα3LN-HSA were fully protected against lethal T. cruzi challenge by inducing a strong anti-α-Gal antibody-mediated humoral response. Furthermore, Galα3LN-HSA-vaccinated α1,3GalT-KO mice exhibited significant reduction (91.7–99.9%) in parasite load in all tissues analyzed, cardiac inflammation, myocyte necrosis, and T cell infiltration. This is a proof-of-concept study to demonstrate the efficacy of a prophylactic α-Gal-based glycovaccine for experimental acute Chagas disease. Chagas disease: Unexplored glycans make for an effective anti-parasitic therapy A vaccine candidate derived from an immunodominant parasitic glycan could offer a much-needed preventive therapy for Chagas disease. The disease, caused by the parasite Trypanosoma cruzi, is endemic to Latin America and an emergent threat to North America and Europe. Current therapies are few, poorly efficacious, and toxic. Igor Almeida, from the University of Texas at El Paso, United States, and his team created a candidate which presents a host with T. cruzi surface-derived α-galactose-containing (α-Gal) glycan covalently linked to a carrier protein. Parasite-derived α-Gal-containing proteins are known to be highly immune-stimulating to humans but were previously unexplored as prophylactics. In a mouse model designed to mimic the human response to Chagas disease, vaccinated animals had a strong antibody response and were fully protected against lethal exposure to T. cruzi. The results offer a promising candidate for future research and validate the method used in this proof-of-concept study. [ABSTRACT FROM AUTHOR]