Date of Award

2022-08-01

Degree Name

Doctor of Philosophy

Department

Chemistry

Advisor(s)

Katja Michael

Abstract

Trypanosoma cruzi, the causal agent of Chagas disease (CD), currently affects 6-8 million people worldwide, predominantly in Latin America, where the parasite is endemic. The treatment of CD is based on two drugs, benznidazole (BZN) and nifurtimox (NFX). These drugs cause adverse effects and are more effective in the acute phase of the disease. Conventional serology assays can be used for diagnosis, but they are not suitable for determining chemotherapy effectiveness because patients take approximately 10-20 years to exhibit negative seroconversion. Although CD has become a global health problem, specific biomarkers are still needed for the follow-up of chemotherapy and vaccine development. T. cruzi has a complex glycocalyx in which glycosylphosphatidylinositol (GPI)-anchored glycoproteins (tGPI-MUC) are prominent. These GPI-MUCs are heavily O-glycosylated with oligosaccharides that are mostly branched. Many of them have ��-Gal moieties at the non-reducing end and are highly immunogenic to humans. ��-Gal residues are the principal targets of protective T. cruzi-specific anti-α-Gal antibodies present in the sera of acute and chronic CD (CCD) patients. Notably, these anti-��-Gal antibodies are known to seroconvert relatively quickly, i.e., within approximately 3-5 years. Therefore, these antibodies are attractive not only for serological diagnosis of CD but also for determining treatment efficacy. The use of tGPI-MUCs as biomarkers (BMKs) to target anti-��-Gal antibodies is less suitable due to the laborious cultivation of parasites, extraction and purification of entire tGPI-MUCs, and inhomogeneity of the material. Instead, utilizing small and homogeneous epitopes (including multiple copies thereof on an inert platform) to which anti-��-Gal antibodies of CD patients respond specifically may be a better approach. Our group has broadly used neoglycoproteins (NGPs) as an alternative for discovering BMKs for diagnosis of CD, early assessment of chemotherapeutic efficacy, and vaccine development. This approach eventually can replace purified T. cruzi lysates as antigens in conventional serological assays. Here we present the synthesis of novel immunogenic oligosaccharides believed to exist on the tGPI-MUC. We synthesized partial structures of potential T. cruzi cell-surface glycans and evaluated their antigenicity using CCD patient sera according to structural information from glycomics data. Chapter 1 briefly introduces Chagas disease and its causal agent, the protozoan parasite T. cruzi. Chapter 2 describes the modular synthesis of three complexes ��-Gal-containing oligosaccharides believed to exist in the tGPI-MUC of T. cruzi. This includes two tetrasaccharides (G36S)2 and (G37S)2 and one pentasaccharide (G35S)2. It will also address their conjugation to BSA, preliminary data about the antibody response using sera from CCD patients, and the effect of glycan loading on the antibody reactivity. Chapter 3 discusses the optimized synthesis of ��-Gal containing trisaccharide (G24S)2 and the development of the conjugation procedure to synthesize neoglycoproteins (NGPs) using different carrier proteins (i.e., human serum albumin (HSA), ovalbumin (OVA), and tetanus toxoid (TT) proteins) and linkers of different lengths (i.e., SIA and SMCC linkers). Finally, chapter 4 features the discovery of a Gal��(1,2)[Gal��(1,6)]Galβ-containing NGP as a synthetic biomarker for CD. It will show the suitability of NGP11b as a biomarker for CCD diagnosis and chemotherapy follow-up by analyzing its reactivity with pooled sera from CCD and healthy individuals.

Language

en

Provenance

Received from ProQuest

File Size

234 p.

File Format

application/pdf

Rights Holder

Elisa Garcia Carvajal

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