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Title: A study of immune responses to Leishmania mexicana antigens and immunogenicity of L. donovani Centrin-3
Author: Asteal, F.
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2011
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Leishmaniasis is a parasitic protozoal disease affecting humans and animals with phlebotomine sand flies as intermediate vectors. The parasite infects phagocytic cells of the mammalian host where they transform from the flagellated promastigote to non-flagellated amastigote phase. There is no effective vaccine in use against this parasite and production relies on finding potent immunogenic antigens with a Th1 bias and long lasting memory response. In this study the immunogenicity of L. mexicana Soluble Leishmania Antigens (SLA) prepared by two different methods (SLA1&2) was investigated by immunisation of Balb/c mice and challenge with live L. mexicana and an in vitro immunological analysis. Immunisation of Balb/c mice with SLA mixed with IFA adjuvant significantly protected against challenge with live L. mexicana parasites. The SLA2 was also further fractionated into six sub fractions by fast protein liquid chromatography (FPLC) using Mono Q columns and the immunogenicity of each fraction was analysed either by ability to stimulate CTL activity against dendritic cells (DCs) target cells loaded with SLA2 and SLA2 fractions or by tritiated thymidine uptake proliferation assay. Immunisation of Balb/c mice with whole SLA as well as the SLA2 fractions induced a significant CTL activity, but responses were higher for the whole SLA. Splenocytes stimulated in vitro for 7 and 14 days with SLA2 and SLA2 fractions induced significant proliferation responses which was increased when splenocytes were stimulated with DCs loaded with these antigens. Leishmania parasites require a number of immune-evasion mechanisms to resist phagolysosome fusion and prevent activation of more-potent acquired immune responses. Down regulation of MHC class I and II expression on infected phagocytic cells may be one of the immune evasion strategies used by the Leishmania parasite. In this study the effect of L. mexicana infection on the expression of surface molecules was investigated in DCs. Unlike treatment with autoclaved parasite, infection of DCs with live L. mexicana parasite down regulated the expression of MHC class I, class II, CD11c, CD80 and CD40. Also, in vitro treatment of DCs with fungizone as early as 1 hour after the initiation of infection with L. mexicana restored their MHC class I expression, as determined by antibody staining and flow cytometry analysis. Interestingly treatment of L. mexicana infected DCs with fungizone also restored their susceptibility to CTL activity. As part of searching for new Leishmania antigens of a potential vaccine application, the immunogenicity of L. donovani centrin-3 (Ldcen-3) was investigated in a Balb/c model. The immunogenicity of Ldcen-3 has not previously been investigated. Ldcen- 3 is a calcium binding protein that has been shown to be involved in duplication and segregation of the centrosome in higher and lower eukaryotes. The Ldcen-3 gene was cloned in various vectors and coated on gold particles for gene gun immunisation. Significant protection was induced by immunisation with 1μg DNA of pcDNA3.1- Ldcen-3 or pCRT7/CT-TOPO-Ldcen-3 constructs. Protection against challenge with live parasite was vector dependent where better protection was induced by pCR T7/CT-TOPO-Ldcen-3. Splenocytes from Balb/c mice immunised with pcDNA3.1- Ldcen-3 or pCRT7/CT-TOPO-Ldcen-3 has a potent CTL response against DC targets loaded with SLA or tumour cells transfected with Ldcen-3 plasmid construct. Collectively, results presented in this study suggest that the whole SLA was more immunogenic than any SLA fractions produced by fast protein liquid chromatography. Results also suggest that L. mexicana could use down regulation of MHC I as a possible mechanism to evade killing by CTL and susceptibility to CTL could be restored by treatment of infected cells with fungizone. These findings also suggest the potential benefit of combination therapy in controlling Leishmania infection. This study has also investigated for the first time the immunogenicity of Ldcen-3 gene which was shown to be highly immunogenic via protection against challenge with live parasite and induction of CTL in immunised mice.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available