Date of Award

2020-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Igor C. Almeida

Abstract

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 or cryptic 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 biomarkers, and vaccine candidates. Here, we investigate the potential of α-Gal-containing glycotopes in the chemotherapeutic follow-up for chronic patients being treated for ChD. We first demonstrate the high affinity of both ChD human serum pool (ChHSP) and purified Ch anti-α-Gal antibodies to the Galα3LN-containing neoglycoprotein (NGP) when compared to normal human serum pool (NHSP) and purified NHS anti-α-Gal antibodies. Subsequently, we show 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. We first vaccinated α1,3-galactosyltransferase-knockout (α1,3GalT-KO) mice, which mimic the human immune response to α-Gal glycotopes, with Galα3LN-HSA and tested its efficacy with a lethal T. cruzi challenge. The Galα3LN-HSA vaccinated α1,3GalT-KO mice were fully protected as shown 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. Finally, we assessed the efficacy of synthetic glycan- and peptide-based vaccine formulations, namely NGP24h and MASPpep-KLH. The former encompasses the trisaccharide Galα1,3Galβ1,4GlcNAcα (Galα3LNα), an immunodominant glycotope found on the trypomastigote mucins, coupled to a maleimide-modified human serum albumin as carrier protein. MASPpep-KLH is composed of a 20-mer peptide (DAENPGGEVFNDNKKGLSRV) found in some members of the mucin-associated surface protein (MASP) of T. cruzi trypomastigotes, coupled to keyhole limpet hemocyanin as carrier protein. MASPpep encompasses B- and T-cell epitopes revealed by proteomics and immunoinformatics. Both vaccines were previously shown to induce considerable protection against acute T. cruzi infection in mice. In this study, we have used Papio hamadryas baboons (Old-World nonhuman primates) as the experimental model, which closely mimics the human immune system. Briefly, baboons vaccinated with NGP24h, MASPpep-KLH, or the combination thereof (NGP24h+MASPpep-KLH) exhibited high levels of IgG and IgA antibodies, and significantly lower inflammation and parasite load in the heart tissue (37%-73% reduction for NGP24h, 78%-93% reduction for MASPpep-KLH, and 41%-71% in the combined NGP24h+MASPpep-KLH). In summary, these are the first rationally designed, synthetic experimental glycan- and peptide-based vaccines against acute T. cruzi infection that have been tested on the nonhuman primate model. This is a proof-of-concept study to demonstrate the efficacy of α-Gal-containing NGPs as biomarkers for the follow-up chemotherapy, and a prophylactic α-Gal-based glycovaccine for experimental acute Chagas disease.

Language

en

Provenance

Received from ProQuest

File Size

139 pages

File Format

application/pdf

Rights Holder

Susana Portillo

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