Masters Degrees (Medical Virology)

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Browsing Masters Degrees (Medical Virology) by Subject "AIDS vaccines"

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    The characterisation and expression of HIV-1 subtype C gag
    (Stellenbosch : Stellenbosch University, 2002) Sampson, Candice Corene; Van Rensburg, E.J.; Engelbrecht, S.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Division of Medical Virology.
    ENGLISH ABSTRACT: The gag gene of HIV-1 encodes for one of the major structural proteins, which contains several conserved cytotoxic T cell (CTL) epitopes. Gag specific CTL responses are important in controlling viral load during acute infection and asymptomatic stages of the infection. Currently, only one complete South African HIV-1 subtype C gag sequence has been published. The first aim of this study was to characterise the complete gag gene of 15 HIV-1 subtype C isolates, to be used as a set of reference sequences in the design of a South African HIV-1 subtype C vaccine. Fifteen HIV-1 subtype C isolates selected for this study, were isolated during 1998 and 1999 from the HIV-1 positive patients attending the Infectious Disease Clinic at Tygerberg Hospital. The gag gene of these isolates was amplified by PCR, cloned into mammalian expression vectors and sequenced. Restriction digest analyses as well as phylogenetic analyses were performed on the sequencing data. Previously published mutational analyses and CTL epitopes were compared to the predicted amino acid sequences of the gag clones. Sequences of 23 complete gag genes representing the 15 HIV-1 subtype C isolates as well as one complete sequence of an HIV-1 subtype B isolate were compiled. Subtyping by restriction fragment length polymorphism (RFLP) would have correctly identified 14 of the 15 subtype C isolates as subtype C and one as unidentifiable. The subtype B isolate would have also been correctly identified. Phylogenetic analyses showed that our subtype C isolates clustered with reference subtype C strains from various countries, including Botswana, India, Israel, Tanzania and Zambia. Strains from Ethiopia and Brazil formed a separate subtype C cluster. The diversity between our isolates was comparable to the diversity seen between all the HIV-1 subtype C strains. Comparisons of previously published mutational analyses and CTL epitopes to the predicted amino acid sequences of the gag clones, showed conservation in most of the clones throughout the sequence. A second aim was to establish transfection and Western Blot techniques in our laboratory for use in future studies. An in vitro transcription! translation assay was performed on the gag clones and the protein producing clones were used to transfect mammalian cells using electroporation. A Western blot was then used to screen for Gag protein expression in the transfected cell Iysates. The in vitro transcription! translation assay showed that seven of the 23 clones could produce a protein of -55 kDa in size. Four out of the seven of these clones gave a weak expression of a-55 kDa protein after transfection in a mammalian cell line. Since the completion of the experimental work of this study, other cloned HIV-1 genes have successfully been transfected into mammalian cells using the electroporation technique and the proteins produced were screened for by Western blot. To conclude with; the native form of the gag gene does not elicit strong expression of the protein, but studies have shown that expression can be improved by sequence-modification of the gag nucleotide sequence. Due to the conservation of gag, the sequence of any subtype C strain can be used for the development of a Southern African vaccine.
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    Characterisation of the HIV-1 subtype C Env gene and the expression of the Env protein from selected isolates in mammalian cells
    (Stellenbosch : Stellenbosch University, 2003-03) De Villiers, Tania; Janse van Rensburg, E.; Engelbrecht, S.; Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Pathology.
    ENGLISH ABSTRACT: At the end of 2002, human immunodeficiency virus (HIV) had infected 42 million people worldwide. The morbidity and mortality rate, as well as the epidemic proportions of the disease have led to concentrated scientific efforts to reveal the disease's pathogenesis and develop effective preventative and treatment measures. Advances have been made to inhibit viral replication by suppressing the virus' ability to replicate by developing antiretroviral treatments, although development of a save and effective vaccine is the only way to stem the pandemic. Advances in vaccine design, animal models and clinical research have led to the creation of promising candidate vaccines to counter this rampage, but most of these vaccines entering phase I-III clinical trials are based mainly only subtype B genomes. HIV-1 subtype C is the most commonly transmitted subtype worldwide, and is the predominant subtype in India, China, East and Southern Africa. A subtype C vaccine is critical for the developing nations such as South Africa, where antiretroviral therapies are largely unaffordable. The envelope gene (env) is an attractive target as immunogen to be included in a HIV vaccine. The envelope protein (Env) elicits neutralising antibodies and cytotoxic T-Iymphocyte (CTl) responses. This protein will therefore be useful in creating a humoral and cellular immune response in the host. A shortage in characterised subtype C env gene sequences from South Africa was recognised, and this study focussed on the characterisation of generated sequences, as well as the expression of selected env genes. These immunogens were created for possible use in a prime-boost vaccine modality. The env genes from recent circulating strains in South Africa were amplified by polymerase chain reaction (PCR). The genes were then cloned for sequencing and expression purposes. Phylogenetic relationships were determined by comparing the sequences to reference subtype strains and subtype C strains. Expression of the genes was assessed by Western Blot in 293 cells with HIV- 1 positive patient sera. Sequence analysis showed a more conserved third variable (V3) loop in South African subtype C sequences, with a more variable region downstream from the loop. The crown sequence (GPGQ) and positions of uncharged or negatively charged residues in the V3 loop indicated a non-syncytium-inducing (NSI) phenotype for the isolates. Phylogenetic analysis showed the sequences to all belong to the C subtype, and further that the sequences were not recombinant, which was confirmed by recombination analysis. The intersample diversity observed for strains from South Africa was significantly higher than distances observed to the subtype C consensus sequence. The South African sequences were distributed across several subclusters in a subtype C phylogenetic tree, highlighting the concept that these infections represent a more longstanding epidemic with multiple introductions from different geographic areas. Western Blot with HIV-1 positive patient sera showed the expression of uncleaved gp160 Env proteins, which were Rev dependent. This study has generated much needed subtype C South African env gene sequences that can be used as basis for modification for use as immunogens in a South African vaccine.
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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.