Leishmaniasis is a disease caused by infection with the obligate intracellular parasite of the genus Leishmania. Leishmaniasis is a public health problem; it is estimated that approximately 12-15 million people worldwide are infected. An estimated 1.5-2 million new cases occur each year, 350 million people are at risk of infection, and it causes 70000 deaths per year.Therefore, development of a vaccine to prevent infection is required. The overall aim of this study was to develop a vaccine to protect against Leishmania infection using live nonpathogenic L. tarentolae transfected with gamma glutamylcysteine synthetase (γGCS) from three different species (L. donovani, L. mexicana and L. major).Previous studies for expressing recombinant γGCS using E. coli as an expression system have shown that it was not possible to produce pure full-length recombinant γGCS protein. Therefore, in this project, using an expression vector that is phylogenically more like Leishmania. L. tarentolae has been used as an expression system for eukayotic recombinant proteins.Studies of the expression of L. donovani, L. mexicana and L. major γGCS recombinant proteins showed that parasites integrated with a green fluorescent protein γGCS-His gene gave stable expression of the fusion protein for six months without the requirement for antibiotics to maintain expression of the gene insert. Supplementing the culture medium with hydrogen peroxide (H2O2) increased proliferation of parasites in cell culture.The addition of a 12 μM of H2O2 supplement increased the number of the parasites in cell culture, which can conclude that expression of the GFP-γGCS-His recombinant protein has been increased.Expression of the recombinant GFP-γGCS-His protein produced full-length protein and truncated protein, but after isolation from an affinity column, it was impossible to produce pure full-length protein for all three transfected parasites. Therefore, this purification method failed to remove non-specific protein contamination.The live non-pathogenic lizard parasite, L. tarentolae, expressing elected Leishmania antigens has recently provided a promising new approach as a safe and effective live vaccine candidate to prevent Leishmaniasis. Here, this study evaluated the immunoprotective potential of a live vaccine against L. major infection in BALB/c mice, using L. tarentolae transfected with the GFP-γGCS-His sequence gene from one of three different species (L. donovani, L. major or L. mexicana) or a ‘triple vaccine’ using a 1:1:1 mixture of L. tarentolae transfected with the GFP-γGCS-His sequence gene of the three pathogenic species.Vaccination with transfected L. tarentolae with GFP-γGCS-His gene from L. donovani, L. major and L. mexicana (triple vaccine) induced significant parasite specific Th1 immune responses based on antibody titres and cytokine production in vitro in stimulated splenocytes and popliteal lymph nodes from immunised mice.Vaccination by subcutaneous injection with the triple vaccine caused the highest percentage reduction in parasite burdens compared to controls ± SE, was 94% ± 0.01 in L. major infected mice. Vaccination with L.t L. maj GFP-γGCS-His, L. mex GFP-γGCS-His and L. don GFP-γGCS-His parasites failed to give significant protection against L. major infection, but vaccination with L. t L. maj GFP-γGCSHis resulted in an 86% ± 0.01 suppression in parasite burden compared to controls.In conclusion, the results of this study indicate that vaccination with transfected L. tarentolae parasites against L. major infection enhanced the protective efficacy and that the triple vaccine is a potential vaccine candidate.
|Date of Award||29 Mar 2018|
- University Of Strathclyde
|Supervisor||Katharine Carter (Supervisor) & Alexander Mullen (Supervisor)|