UBC Theses and Dissertations
Protection against leishmaniasis by DNA injection of a plasmid encoding the Leishmania major Cpn60 gene Webber, Jane
The leishmaniases are parasitic protozoan infections caused by members of the genus Leishmania. Injection of Leishmania major into various strains of inbred mice cause disease symptoms which mimic the symptoms of a human leishmanial infection. Protection against an L. major infection in BALB/c mice depends on the generation of a macrophage-activating CD4 ⁺ Th1 immune response with production of IL-2 and IFNγ, but not IL-4, a Th2 cytokine that results in disease exacerbation. Immunization using DNA vaccines is a promising new approach for the prevention of diseases caused by intracellular pathogens. The antigen of interest encoded in the plasmid is transcribed and translated by the host cellular machinery and presented to the immune system inducing specific cell-mediated and humoral immune responses against the encoded protein. In this study a eukaryotic expression vector, pcDNA3.1 encoding the L. major chaperonin 60 (Cpn60) was constructed and tested in vitro to confirm that the mammalian translational machinery could synthesize the protein. Cell-free coupled transcription/translation using rabbit reticulocyte lysate and transfection studies using COS-7 cells were carried out by SDS-PAGE and western blot analysis of protein products. A single protein of the correct size was produced in vaccine samples but not in the Controls. To test the vaccine efficacy, BALB/c mice were immunized with either the vaccine construct, empty plasmid control or PBS, and boosted twice two weeks apart. Spleen cells from mice immunized with the vaccine construct did not produce significant amounts of the Thl cytokines interleukin-2 (IL-2) or gamma interferon (IFN-γ), or the Th2 cytokine IL-4, when cultured with soluble Leishmania antigen (SLA) in vitro. Sera from each mouse group was pooled and used to probe immunoblots where protein from control organisms, purified human Cpn60 and L. major total protein was used as antigen. Sera from the vaccine mouse group did not detect the L. major Cpn60 protein. The results of these experiments indicated that the protein was expressed in vitro but immunization experiments were unsuccessful using this vaccination protocol. Review of vaccination techniques and a change in the immunization site may improve chances for a successful outcome.
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