A dissertation submitted to Faculty of Health Sciences, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science in Medicine Johannesburg, June 2016 / Human Leukocyte Antigen (HLA) class I (HLA-I)-restricted CD8+ cytotoxic T lymphocyte (CTL) responses are major drivers of human immunodeficiency virus type 1 (HIV-1) evolution, resulting in the selection of CTL escape mutants. This is particularly well described for HIV-1 proteins such as Gag, Pol and Nef. For the accessory proteins Vif, Vpr and Vpu though, it is still not clear to what extent HLA-I-mediated responses influence their evolution. Moreover, preliminary data from our laboratory showed that Vpu was the most targeted of the accessory proteins with regards to CD8+ CTL and natural killer (NK) cell responses in a long term non progressor/elite controller cohort. Thus, the overall aims of the study were to describe immune escape in Vif, Vpr and Vpu epitopes from HIV-1 infected patients in South Africa, and to clone, express and purify recombinant Vpu for the ultimate detection of anti-Vpu antibodies in South African patient sera. Longitudinal plasma samples were available for 25 patients from two cohorts (18 from Lung and seven from M002). Viral RNA was extracted and the vif, vpr and vpu regions were RT-PCR (Reverse transcription polymerase chain reaction) amplified and sequenced. HLA-A, B and C data for Lung cohort individuals were available, thus the HLA-A, B and C alleles were typed and assigned for the M002 cohort individuals. The Vif, Vpr and Vpu amino acid sequences were subsequently extensively analysed for HLA-driven CTL escape mutations. Lastly, three vpu sequences, Cons. C, 05ZAFV05 and 05ZAFV15 were selected for codon-optimization (humanized), each cloned into the pcDNA3.1/V5-His mammalian expression vector, and used to optimize expression of recombinant Vpu proteins in HEK 293T cells. The HEK 293T cells were harvested and the Vpu was purified using standard nickel affinity chromatography procedures. Protein expression and purification was monitored by SDS-PAGE, Western blot and dot blot analyses. Overall, longitudinal sequences were obtained from Vif, Vpr and Vpu from eight of the 18 patients in the Lung cohort and all seven patients in the M002 cohort. Of these, HLA-driven immune escape mutations were detected in four patients from the Lung cohort and six from the M002 cohort. Interestingly, for the M002 cohort, some reversions to the baseline sequences were noted over time. None of the identified escape mutations are amongst the best described and most optimal HIV-1 CD8+ CTL epitopes of HIV-1 accessory proteins in the HIV-1 databases. Eight and eleven HLA alleles contributed to immune escape in the Lung and M002 cohort Vif, Vpr and/or Vpu sequences, respectively. Overall, HLA-driven immune pressure in the Vif, Vpr and/or Vpu motifs described here may have contributed to the changes in viral loads seen in these patients over time, and likely impacted iii on disease progression, albeit in combination with escape mutations in the more highly targeted HIV-1 proteins, such as Gag, Pol and Nef. Future work looking at longitudinal sequence analysis of the entire HIV-1 proteome in these patients will likely reveal the contributing factors leading to immune escape and ultimately impact disease progression to AIDS. Expression of all three recombinant Vpu proteins in HEK 293T cells was successfully optimized however, as evidenced by SDS-PAGE, Western blot and dot blot analyses, the expression and purification protocol used in this study did not provide sufficient protein yields, nor an optimally purified protein for use in the downstream assays required. Future work will focus on optimization of Vpu expression and purification protocols to aid in the detailed elucidation of the role of Vpu and anti-Vpu immune responses in control of HIV-1 infection. / MT2016