Trypanosoma species research and implications (3)Glycosylation and Glycoproteins Research (2)Carbohydrate Chemistry and Synthesis (2)Escherichia coli research studies (1)Lysosomal Storage Disorders Research (1)
Balouz V, Lopez R, Conte N, Abal M, Ducrey I, et al.
PLoS neglected tropical diseases •
Chagas disease (ChD), caused by the parasitic protozoan Trypanosoma cruzi, is a lifelong, neglected tropical disease with substantial medical and socioeconomic impact. Despite this situation, currently available diagnostic and therapeutic methods display serious limitations. A promising strategy to improve ChD serodiagnosis involves targeting parasite carbohydrate antigens, particularly the α-galactosyl-rich mucins that coat the surface of bloodstream trypomastigotes (tGPI-mucins). Here, we present a concise and efficient protocol for the chemical synthesis of a tGPI-mucin-derived glycotope, the disaccharide α-D-Galp-(1 → 3)-β-D-Galp, and its functional conjugation to different scaffolds using the squarate method. A neoglycoprotein made upon a bovine serum albumin (BSA) carrier decorated with ∼38 units of the disaccharide, termed BSA-Di, was interrogated with sera of chronic ChD patients and healthy individuals from Argentina using an in-house enzyme-linked immunosorbent assay (ELISA). BSA-Di exhibited excellent sensitivity and effectively discriminated between ChD-positive and negative sera with high accuracy (AUC = 0.905), though its specificity was partially affected by cross-reactivity of some non-ChD sera containing natural α-Gal antibodies. Conjugation of α-D-Galp-(1 → 3)-β-D-Galp to T. cruzi antigenic peptides, instead of BSA, corroborated these findings and enabled the generation of bivalent ChD diagnostic reagents combining glycan- and peptide-based epitopes. Overall, our results identify α-D-Galp-(1 → 3)-β-D-Galp as a robust and reliable biomarker of T. cruzi infection. The methodologies and tools described here, together with optimized derivatives, are expected to positively impact ChD serological applications.
Abal M, Balouz V, Lopez R, Giorgi ME, Marino C, et al.
PLoS neglected tropical diseases •
Proper evaluation of therapeutic responses in Chagas disease is hampered by the prolonged persistence of antibodies to Trypanosoma cruzi measured by conventional serological tests and by the lack of sensitivity of parasitological tests. Previous studies indicated that tGPI-mucins, an α-Gal (α-d-Galp(1→3)-β-d-Galp(1→4)-d-GlcNAc)-rich fraction obtained from T. cruzi trypomastigotes surface coat, elicit a strong and protective antibody response in infected individuals, which disappears soon after successful treatment. The cost and technical difficulties associated with tGPI-mucins preparation, however, preclude its routine implementation in clinical settings. We herein developed a neoglycoprotein consisting of a BSA scaffold decorated with several units of a synthetic α-Gal antigenic surrogate (α-d-Galp(1→3)-β-d-Galp(1→4)-β-d-Glcp). Serological responses to this reagent, termed NGP-Tri, were monitored by means of an in-house enzyme-linked immunosorbent assay (α-Gal-ELISA) in a cohort of 82 T. cruzi-infected and Benznidazole- or Nifurtimox-treated children (3 days to 16 years-old). This cohort was split into 3 groups based on the age of patients at the time of treatment initiation: Group 1 comprised 24 babies (3 days to 5 months-old; median = 26 days-old), Group 2 comprised 31 children (7 months to 3 years-old; median = 1.0-year-old) and Group 3 comprised 26 patients (3 to 16 years-old; median = 8.4 years-old). A second, control cohort (Group 4) included 39 non-infected infants (3 days to 5 months-old; median = 31 days-old) born to T. cruzi-infected mothers. Despite its suboptimal seroprevalence (58.4%), α-Gal-ELISA yielded shorter median time values of negativization (23 months [IC 95% 7 to 36 months] vs 60 months [IC 95% 15 to 83 months]; p = 0.0016) and higher rate of patient negative seroconversion (89.2% vs 43.2%, p < 0.005) as compared to conventional serological methods. The same effect was verified for every Group, when analyzed separately. Most remarkably, 14 out of 24 (58.3%) patients from Group 3 achieved negative seroconversion for α-Gal-ELISA while none of them were able to negativize for conventional serology. Detailed analysis of patients showing unconventional serological responses suggested that, in addition to providing a novel tool to shorten follow-up periods after chemotherapy, the α-Gal-ELISA may assist in other diagnostic needs in pediatric Chagas disease. The tools evaluated here provide the cornerstone for the development of an efficacious, reliable, and straightforward post-therapeutic marker for pediatric Chagas disease.