Masters Degrees (Medical Virology)

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Browsing Masters Degrees (Medical Virology) by Author "Fiff, Fabian"

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    Transfection of baboon dendritic cells with plasmid DNA containing HIV-1C genes : effect of transfection methods on antigen processing and presentation to T lymphocytes
    (Stellenbosch : University of Stellenbosch, 2005-12) Fiff, Fabian; Glashoff, R. H.; Liebrich, W.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Pathology. Medical Virology.
    There is an urgent need for a safe, effective, affordable human immunodeficiency virus type 1 (HIV-1) vaccine that induces both cellular and humoral immunity. A popular strategy for vaccine design is the use of plasmid DNA encoding HIV-1 genes for priming vaccinations followed by either viral vector or recombinant protein boosting. DNA-based vaccines are attractive because they are safe, easily administered and can induce both cellular and humoral immune responses. In order for DNA vaccination to induce a potent immune response it is necessary for plasmid-encoded genes to be targeted to dendritic cells (DCs) as these are the key antigen presenting cells in natural HIV infection. The immunogenicity of all potential vaccine candidates needs to be assessed in animal models prior to entry into human trials. Nonhuman primates are the best alternative to humans for assessment of vaccine immunogenicity and protective efficacy. In order to clearly understand how DNA vaccines interact with DCs, suitable in vitro DC culture systems for nonhuman primates need to be developed. This study investigated the culture and characterisation of chacma baboon DCs in vitro, and was the first to assess the effect of various transfection methods on baboon DC maturation and function. The study also evaluated the efficacy of a candidate HIV-1 subtype C DNA vaccine at the level of baboon DC transfection, gene transcription and antigen presentation. Generation of immature DCs (iDCs) in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF) was accompanied by a loss in the monocyte marker CD14. Expression of the markers CD80 and CD83 was observed on a minority of iDCs, whereas CD86 was expressed on almost all iDCs. Following maturation, all these markers were expressed on an increased number of cells, a pattern of marker expression and upregulation that is similar to that observed in both human and macaque DCs. Transfection of baboon DCs by passive pulsing, lipofection and electroporation was evaluated and compared in several ways. Transfection efficiency, cytotoxicity, the effect of the transfection on DC maturation and subsequent presentation of plasmidencoded antigen to memory T lymphocytes was examined. Baboon DCs lipofected with pDNA efficiently took up HIV-1 subtype C plasmid DNA, transcribed plasmid-encoded genes into mRNA, translated the mRNA into protein, processed the protein and presented peptide antigens to antigen-specific memory T cells. The other methods of transfection were less effective than lipofection due to either decreased transfection efficiency or increased cell cytotoxicity. However, neither lipofection nor passive pulsing in any way negatively impacted on DC marker, CD83, or costimulatory molecule, CD80 and CD86, upregulation. Both methods were found to be as effective as a standard cytokine maturation cocktail in inducing DC maturation. Transfected DCs were also found to be more potent inducers of allogeneic T cell stimulation than their untransfected counterparts, which would appear to indicate enhanced major histocompatibility complex (MHC) expression concurrent with DC maturation marker expression. Lipofection with candidate HIV-1 subtype C vaccine plasmid DNA constructs led to antigen-specific expansion of autologous memory T cells, a finding which indicates the effective expression of plasmid-encoded HIV genes in baboon DCs. This study highlights the functional activity of in vitro generated baboon DCs and provides the groundwork for future studies addressing targeting of plasmid DNA to DCs and enhancement of expression of plasmid-encoded antigens in DCs. A more detailed evaluation of baboon DC interaction with simian immunodeficiency viruses/chimeric simian human immunodeficiency viruses (SIVs/SHIVs) may also reveal how the course of infection in this primate differs from that seen in the macaque or chimpanzee and also how it relates to HIV-1 infection in humans.