Characterisation of the anti-Gal response to Leishmania infection, and its possible exploitation for the diagnosis of cutaneous leishmaniasis

The surface glycocalyx of Leishmania parasites consists of a complex mix of glycoconjugates, with varied roles in cell invasion and protection from host immune responses. Most abundant amongst these is a family of glycoinositolphospholipids (GIPLs), the glycan components of which vary between Leishmania species. Type-II GIPLs are abundantly expressed by L. major parasites and contain terminal αGalactosyl residues that are recognised by serum from infected patients. Since the oligosaccharidic portion of the main L. major GIPLs terminate with the linear sequences Galα(1,3)Galfβ(1,4)-R (GIPL-2) or Galα(1,6)Galα(1,3)Galfβ(1,4)-R (GIPL-3), which differs to the main epitope (Galα(1,3)Galβ(1,4)GlcNAcβ1-R) recognised by normal anti-Gal antibodies, I hypothesised that the specificity of the anti-Gal produced during a course of a Leishmania infection will be different to that of normal anti-Gal. In this thesis, I set out to characterise the human anti-Gal response that is triggered during Leishmania infection, aiming to exploit this knowledge for the development of a novel diagnostic test. The utility of native GIPLs extracted from both L. major and L. tropica cells as diagnostic antigens was assessed first through a combination of Thin Layer Chromatography (TLC) immuno- and lectin-staining, mass spectrometry analysis, and chemiluminescent ELISA (CL-ELISA). GIPLs extracted from L. major promastigotes were strongly recognised by sera from L. major-positive patients, but not from L. tropica-positive patients, or that from healthy controls. In addition, it was confirmed that L. tropica does not express αGalactosylated GIPLs, suggesting that this residue must be decorating an uncharacterised glycoconjugate. To determine the specificity of the Leishmania spp. anti-Gal, a bespoke panel of neoglycoproteins (NGPs) was used to investigate anti-Gal produced in response to leishmaniasis infection in three patient cohorts. These sample collections encompassed the spectrum of the leishmaniases, from systemic visceral disease to localised cutaneous lesions. Antibody detection in Leishmania patients has potential in screening of suspect cases, but also in monitoring response to treatment. NGPs with Galα(1,3)Galfβ-R and Galα(1,6)Galα(1,3)Galfβ-R terminating glycans were the best discriminators of L. major infections and also showed potential in the diagnosis of American cutaneous leishmaniasis caused by L. braziliensis. In addition, the presence of a terminal β-galactofuranose residue in these NGPs dramatically improved their recognition in CL-ELISA, and their value in the diagnosis of Old World CL. Combining these two glycotopes maximises antibody detection, through capture of two anti-Gal populations. Fluorescent microscopy analysis confirmed L. major cells express αGalactosylated epitopes throughout the parasite lifecycle, whilst in L. tropica the same epitope was only detected in promastigote cells. While the nature of this epitope remains unknown, future work should focus on the analysis of L. tropica glycoconjugates. Results in this thesis are discussed in relation to the variability of αGalactosyl epitopes recognised by sera from patients with different Leishmania infections, and their possible exploitation in the development of novel disease biomarkers.