Date of Award

2017-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Rosa A. Maldonado

Abstract

Protozoan parasites from the genus Leishmania cause broad clinical manifestations known as leishmaniasis, which affect millions of people worldwide. Cutaneous leishmaniasis (CL) is mainly caused by either Leishmania major or L. tropica parasites, which produce localized cutaneous ulcers, often leading to scarring and social stigma. Currently, the disease has reached hyperendemicity levels in the Middle East due to conflict and human displacement and is one the most common forms of the disease in the Old World. Furthermore, the first choice of treatment in that region continues to be pentavalent antimonials, which are costly and highly toxic, and current vector control measures alone are not sufficient to stop disease transmission. CL remains largely neglected, with no prophylactic or therapeutic vaccine available, and existing drug treatments are expensive, have toxic side effects, and resistant parasite strains have been reported. Hence, further therapeutic and preventive interventions against the disease are necessary.

In this regard, previous studies from our laboratory, reported promising in vitro anti-leishmanicidal findings of a novel series of organometallic compounds containing RuII and clotrimazole (CTZ) termed AM160 or AM162 [1]. For the first part of the present study, we determine the efficacy of AM160 or AM162, in BALB/c infected mice with L. major metacyclic promastigotes to alleviate CL. AM162 treatment (4 mg/kg/day) reduced the lesion size in the murine model of CL by a 68%, a markedly better effect than CTZ alone or a RuII complex not containing CTZ (C3). In addition, in a hyper infection experiment, AM162 treatment at 6 mg/kg/day significantly (p=0.0146) reduced CL footpad lesions and parasite load in comparison with vehicle control group, with no significant toxicity observed. Moreover, we investigated the potential mechanism of cell death inflicted by RuII CTZ complexes on L. major promastigotes. AM160 and AM162 exhibited pro-apoptotic-like properties, implicating phospholipids externalization, mitochondrial depolarization and DNA fragmentation, as its mechanism to induce cell death in L. major parasites; encompassing early and late hallmarks of apoptosis. Overall, these findings indicate that AM162 complex is an efficient compound for the treatment of CL in a murine model.

Furthermore, the second part of this Dissertation focuses on the development of a vaccine based on terminal, non-reducing, and linear α-galactopyranosyl (α-Gal) epitopes that are abundantly found on the plasma membrane glycolipids of L. major known as glycoinositolphospholipids (GIPLs). Previous studies have suggested that sugars are promising vaccine candidates against leishmaniasis, since most parasite species have a cell surface coat composed of immunogenic sugars, including linear α-Gal epitopes, which are absent in humans. 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 (�1,3GalT-KO) mice. These transgenic mice, similarly to humans, do not express non-reducing, linear �-Gal epitopes in their cells and are, therefore, capable of producing high levels of anti-�-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. When tested in transgenic mice, which like humans, lack �-Gal epitopes in their cells, NGP5B was able to induce a significant partial protection against L. major infection by significantly reducing mouse footpad lesions and parasite burden. Altogether, we propose NGP5B as a promising preventive vaccine, and AM162 as a treatment option for CL caused by L. major.

Language

en

Provenance

Received from ProQuest

File Size

124 pages

File Format

application/pdf

Rights Holder

Eva A Iniguez

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Parasitology Commons

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