Doctoral Degrees (Medical Virology)

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Browsing Doctoral Degrees (Medical Virology) by Author "Botha, Johannes Christiaan"

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    The role of clonally expanded HIV-1 infected cells in maintaining HIV reservoirs in adults and children on antiretroviral treatment
    (2020-12) Botha, Johannes Christiaan; van Zyl, Gert U; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.
    ENGLISH ABSTRACT: The HIV-1 pandemic remains a major public health dilemma. Treatment with combination antiretroviral therapy (cART) can, in the majority of patients, sufficiently suppress HIV viral replication. However, cART is not a cure as it does not affect long-lived reservoirs. HIV proviruses are harboured in the genome of long-lived CD4 helper cells that are maintained by having long half-lives and by being replenished through cellular proliferation. HIV infected cells that proliferate can be identified as clonal populations by all having the same integration site. A small proportion of proviruses are intact from where HIV rebounds after therapy interruption and is the barrier to achieving cure or remission. Therefore, many molecular assays have been designed to characterise HIV proviruses, with a focus on intact proviruses. From this the proviral landscape is estimated to consist of ~2% intact proviruses and up to 98% defective proviruses comprising deletions and hypermutations. However, highly deleted proviruses such as solo-long terminal repeats (LTRs) remain largely uncharacterised. Recently, it has become apparent that some proviral clones are able to express infectious HIV and cause clonal viraemia in patients on cART, which are not indicative of ongoing cycles of viral replication. This has further confirmed the importance of proviral clones in maintaining the HIV reservoir. Subsequently, aspects of proviral clones were investigated in this study. A novel assay capable of characterising severely deleted proviruses by targeting the unique integration sites was developed. The characterisation of proviral clones in paediatric patients with this novel assay revealed that severely deleted solo-LTR proviruses may be more prevalent than previously expected. Further investigation of these solo-LTR proviral clones was performed with another novel assay capable of quantifying proviruses based on unique integration sites. The longitudinal waxing and waning of solo-LTR proviral clones could be observed. Although solo-LTR proviruses do not contribute to the true reservoir, it suggests that current proviral landscape proportions require adjustment to account for all HIV integration events in cells. Clonal viraemia was studied in HIV positive adult patients on long term cART presenting with non-suppressible HIV viraemia. Three clusters of monotypic plasma viraemia and cell associated DNA HIV sequences were identified in one patient. These monotypic proviruses were shown to be replication competent by viral outgrowth. This provides evidence that clonally proliferated cells harbour at least a proportion of intact proviruses and therefore constitute an important component of the true HIV reservoir. Two additional patients were identified with uncommon cases of sustained viraemia. Firstly, a probable large cell clone harbouring non-infectious virus was found to leak proviral nucleic acid into plasma, resulting in apparent treatment failure. Secondly, possible clonal viremia was observed in a patient with very slow decaying viral load, despite objective evidence of adherence and initial undetectability of treatment-relevant drug resistance over a period of 2 years. We conclude that clonal viraemia may be underreported, and that cases like these show that clonal viraemia should be considered as an explanation of non-suppressed viraemia or apparent therapy failure in the management of HIV infected patients on cART.