Background: Cutaneous Leishmaniasis caused by the Leishmania mexicana complex is associated with unpleasant or disfiguring lesions, for which there only limited treatment options. The life cycle of L. mexicana consists of two stages which involve different immune evasion mechanisms: promastigote and amastigote. Understanding parasitic interactions with host cells and developing a protective vaccine could improve the management and treatment of the disease. Aims: • To construct and compare the immunogenicity of 3 Leishmania genes in 3 different plasmids. • Study the effect of long term in vitro culture on the virulency of L. mexicana and its interaction with host cells. • To analyse the influence of Leishmania infection on MHC class I expression by susceptible human cell lines (U937 macrophages, U937 and MonoMac-6 monocytes), and Toll-like receptor, cytokines and chemokines gene expression profiles. Methodology: Three Leishmania genes (L. mexicana GP63, L. donovani centrin1 and L. donovani centrin3) were cloned into three plasmids (pcRT7/CT-TOPO; VR1012; and pcDNA3.1/Hygro(-)). The immunogenicity of the prepared DNA constructs and their empty counterparts was assessed in Balb/c mice using gene gun immunisation method. An in vivo model of attenuated L. mexicana was produced by growing the parasite in vitro for up to passage 20, and testing the infectivity of these parasites in vivo and in vitro. The influence of infecting target cells with virulent and avirulent L. mexicana at different growth stages on MHC class I expression was determined by flow cytometry. Gene expression profiles were determined by qPCR analysis of extracted mRNA. Results: All tested Leishmania DNA constructs were highly immunogenic compared to the controls, as assessed using ELISPOT and cell proliferation assays. A novel survival assay developed in this study illustrated that macrophages derived from immunised mice were resistant to Leishmania infection. The parasite at passage 1 was highly infectious (virulent), but this progressively decreased to be completely avirulent at passage 20. This was associated with a significant down regulation of virulence-associated genes (GP63, LPG2, CPC, CPB2, CPB2.8, CHT1, LACK and LDCEN3) at passage 20, and was also accompanied by morphological changes. The avirulent parasite was unable to transform to the pathogenic amastigote stage in infected target cells. The gene expression profile of toll-like receptors (TLR-1, TLR-2, TLR-4, and TLR-9), cytokines (IL-1, IL-6, IL-10, IL-12β, TNF-α, and TGF-β), and chemokines (CCL-1, CCL-2, CCL-3, CCL-4, CCL-5, and CCL-22) in target cells was were induced and inhibited according to the virulence status of the parasite. Similar and significant down regulation of MHC class 1 was induced by infection of target cells for 24 hours with both virulent and avirulent L. mexicana parasite, however after 48 hours of infection only cells infected with avirulent but not virulent parasite have significantly restored their MHC class I expression. Conclusions: Since no differences in the immunogenicity of the three plasmids encoding the same Leishmania gene were observed, immunogenicity is not dependent on the plasmid type. The failure of the avirulent L. mexicana parasite to infect Balb/c mice, and its inability to produce the pathogenic amastigote stage in vitro suggests that it might have potential as a vaccine candidate. This was also supported by the up regulation of Th2 mediators following the infection with virulent compared avirulent parasites. The level of MHC class I down regulation was dependent on parasite growth stage, virulency and infection dose.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:658375 |
| Date | January 2014 |
| Creators | Ali, K. S. M. |
| Publisher | Nottingham Trent University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://irep.ntu.ac.uk/id/eprint/156/ |
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