Since the advent of antiretroviral therapy (ART) and the resultant suppression of viraemia in the majority of people living with HIV-1 (PLWH) on ART, HIV-1 infection has become manageable and PLWH have similar life expectancies as uninfected persons. However, ART is not curative, is needed lifelong, and its cessation leads to the recrudescence of viraemia. This is due to the formation of a latent reservoir that is long-lived and stable over time, precluding HIV-1 cure. The factors affecting reservoir formation, establishment, and kinetics are not fully understood. Furthermore, differences exist at the population level in disease progression in PLWH depending on ethnicity, biological sex, and infecting viral subtype. Similarly, differences in the latent reservoir of HIV-1 have been described, although less extensively. Understanding what shapes the latent HIV-1 reservoir is critical for developing strategies for cure. Furthermore, it is imperative that cure research is undertaken in diverse populations to ensure coverage of knowledge across different demographics. The latter will ensure that a cure strategy can be developed that will be globally implementable. In the Introductory chapter of this thesis, I provide a detailed review of the current literature and address the need for cure research in low-and middle- income countries. If a global cure is to be achieved, the burden of HIV-1 will need to be addressed in many different populations, most notably African women, as women bear the burden of HIV-1 globally. In South Africa, the country in the sub-Saharan African region with the highest prevalence of HIV-1, women are roughly twice as likely to be living with HIV than men (aged 15 to 49), with a prevalence rate 6% higher than the national average of 19%. Since women are underrepresented in HIV-1 research in general and more specifically in cure studies due to the paucity of research in countries outside of the global North, reservoirs and cure strategies ii need to be characterized in this context. Furthermore, while early treatment is the WHO standard of care for people diagnosed with HIV, a large majority of PLWH only initiated treatment in chronic infection. Since early ART is known to restrict formation of the latent reservoir of HIV-1, research in both early and late ART initiators is necessary. This research focused on characterising the viral reservoir in South African women in a well-established cohort of women who were recruited during acute HIV infection and followed until treatment initiation (which occurred during chronic infection) and beyond. Overall, this thesis focuses on characterising immune activation and inflammation during the course of both untreated and treated HIV-1 infection in a cohort of South African women and subsequently determining whether clinical or immune measures influence characteristics of the latent reservoir of HIV-1. T cell activation and the levels of soluble inflammatory cytokines in plasma were determined in forty-six women in the CAPRISA 002 Acute infection cohort. Chapter 2 describes the cellular immune activation and inflammation profiles of these participants throughout the course of infection at the following timepoints: acute infection, oneyear post-infection, and within a year preceding ART initiation, and two- and four- years postART initiation. T cell activation peaked in chronic infection and reduced dramatically after ART initiation. CD4+ and CD8+ T cell activation reached a post-treatment nadir by two years after ART initiation. Cytokine measures were within the ranges reported in the literature for PLWH. Notably CXCL-10 levels in plasma decreased significantly between two- and four years post-ART, indicating that it may be a sensitive marker of ongoing systemic inflammation in people on ART. In short, the T cell activation and inflammation profiles of the women in this study reflected what has been observed in other cohorts. iii The size of the replication-competent HIV-1 reservoir, measured by quantitative viral outgrowth assay after 5 years of suppressive antiretroviral therapy (ART), was quantified in twenty women of the cohort. In Chapter 3, the clinical and immunological correlates of reservoir size were investigated. Predictive modelling showed that the size of the replicationcompetent reservoir is directly related to viral load and CD4+ T-cell counts over the course of infection, although these measures do not fully predict reservoir size. We found that, in addition to viral load and CD4+ T-cell count, CD8+ T-cell activation within the year preceding ART, nadir CD4+ T-cell count, and baseline as well as on-treatment CD4:CD8 ratio at the time of sizing was associated with replication-competent reservoir size. We provide evidence that the late CD8+ T-cell activation level before treatment, together with viral loads and CD4+ T-cell counts, are directly related to the size of the replication-competent reservoir of HIV-1. Our results are consistent with the hypothesis that the host immune milieu near the time of ART initiation plays an important role in shaping the durable reservoir of HIV infection that persists on ART. Another characteristic of the HIV-1 reservoir is persistence: the presence of all forms of HIV1 within cells and tissues that contribute to pathogenesis, including defective, non-induced, and non-integrated forms of HIV-1. In Chapter 4, total HIV-1 DNA levels were measured as a proxy for viral persistence in thirty-one participants, and the correlates thereof investigated. The HIV-1 DNA levels in this cohort were similar to those reported in the literature for other cohorts where participants initiated therapy in late chronic infection. HIV-1 DNA levels did not differ significantly between two- and four years post-ART, but there was a trend to lower HIV-1 DNA when measuring pol versus gag gene frequencies in peripheral blood mononuclear cells (PBMC). These findings indicate that HIV-1 DNA decay rates may differ depending on the gene being measured, even when using the same assay. A weak significant correlation was iv found between CD4+ T cell counts at ART initiation and the change in HIV-DNA levels between two-and four years on ART. There was a significant correlation between residual CD4+ T cell activation at four years post-ART initiation and gag copies per million PBMC. A trend towards a correlation was found between CD4+ T cell activation and pol copies per million PBMC at the same timepoint. Finally, we found significant correlations between several cytokines at one-year post-infection and within one year pre-ART. These findings further solidify the hypothesis that the immune milieu around the time of ART initiation and after may play a complex role in formation of the viral reservoir of HIV-1. Our studies show a significant link between chronic immune activation and replication competent reservoir size, and also ongoing immune activation and viral persistence on ART. Further studies into whether these immune measures affect the timing of establishment and clonality of the reservoir in this cohort are ongoing and will inform the field about whether differences in cure strategies will need to be explored for those PLWH who had high levels of chronic immune activation before treatment initiation and subsequent shaping for the long-lived viral reservoir.

Includes abstract. / Includes bibliographical references. / Includes bibliographical references (leaves 74-81). / Over-expression of β-arrestin 1 in COS-l cells revealed that the mammalian type GnRH receptor can internalise in a β-arrestin dependent manner whereas the internalisation of the mammalian type I GnRH receptor is β-arrestin independent. investigate which domains on the mammalian type II GnRH receptor are required for β~arrestin dependent internalisation, chimeric receptors were created. / The mammalian type II GnRH receptor possesses an intracellular C-terminal tail that is known to play a role in desensitisation, internalisation and overall signalling in GPCRs. On the other hand, the mammalian type I GnRH receptor, which lacks a C-terminal tail, does not readily desensitise and undergoes slow internalisation compared to the mammalian type II GnRH receptor. Over-expression of ß-arrestin 1 in COS-l cells revealed that the mammalian type GnRH receptor can internalise in a ß-arrestin dependent manner whereas the internalisation of the mammalian type I GnRH receptor is ß-arrestin independent. investigate which domains on the mammalian type II GnRH receptor are required for ß-arrestin dependent internalisation, chimeric receptors were created. Firstly, a chimera in which the full length type II GnRH receptor C-terminal tail was added to the tail-less type I GnRH receptor (TI/T2tail) was created. This chimera internalised in a ß-arrestin and GRK dependent manner, demonstrating that the type II GnRH receptor C-terminal tail confers ß-arrestin JGRK dependent internalisation on the originally ß-arrestin/GRK insensitive GnRH receptor. Mutating the putative GRK and casein kinase phosphorylation sites (serines 338 and 339) on the C-terminal tail of TI/T2tail to alanine residues did not abolish ß-arrestin dependent internalisation but eliminated GRK dependent internalisation, suggesting that other regions on the C-terminal tail are required for ß-arrestin dependent internalisation. A second chimera, in which the whole third intracellular loop of the type II GnRH receptor was replaced with that of the type I GnRH receptor (T2/TIICL3), was created. This chimera could not utilise ß-arrestin in its internalisation, indicating that the third intracellular loop of the type II GnRH receptor is required for ß-arrestin dependent internalisation. An alignment of the amino acid sequences of the two mammalian GnRH receptor third intracellular loops identified a basic residue rich area (R234, R236 and K237) on the type II GnRH receptor that was absent on the type I GnRH receptor. Interestingly, the triple mutant (R234,236,K237 A) still internalised in a ß-arrestin dependent manner, however, truncation of the C-terminal tail of R234,236,K237A abolished the ability of the receptor to internalise in a ß-arrestin dependent manner. This result indicated that the C-terminal tail of the type II GnRH receptor was compensating for the absence of the three basic residues. To summarise, this thesis demonstrates that the C-terminal tail of the type II GnRH receptor can confer ß-arrestin dependent intemalisation on the type I GnRH receptor. Furthermore, the third intracellular loop, and more specifically, basic residues R234, R236 and K237 on the mammalian type II GnRH receptor are required for ß-arrestin dependent intemalisation.

Includes abstract. Includes bibliographical references.

Includes bibliographical references (leaves 108-126). / Infection by the parasitic nematode Nippostrongylus brasiliensis involves migration through the lungs, causing significant damage and generating chronic lung pathology. The resolution of N. brasiliensis infection and also the induction of pulmonary pathology, including goblet cell hyperplasia and acute airway inflammation, depend on IL-4Rá signalling. A key feature of IL-4Rá signalling is the induction of a strong TH2 response which induces the development of alternatively activated macrophages (AAMs). AAMs are associated with tissue remodelling and the control of exacerbated inflammation. In order to investigate potential roles for IL-4Rá in N. brasiliensis' induced lung pathology, we infected mice deficient for IL-4Rá on macrophages and neutrophils (LysMCreIL-4Rá-/lox), IL-4Rá -/- and control mice (IL-4Rá-/lox) with N. brasiliensis and examined lung pathology at days 5, 42 and 180 post infection (p.i.).All three mice strains showed similar emphysemic-like pathology (alveolar dilatation) and airway hyperresponsiveness (AHR) which was well developed by day 42 p.i. and remained chronic. However, LysMCreIL-4Rá-/lox mice consistently demonstrated earlier and increased pulmonary inflammation when compared to IL-4Rá-/lox control mice and IL-4Rá-/- mice. Immunological studies at day 5 p.i. revealed that there were increased CD4+ and CD8+ T-cell numbers and increased CD4+ IL-4 and IL-13 production in the lungs of LysMCreIL-4Rá-/lox mice when compared to control and IL- 4Rá-/- mice. LysMCreIL-4Rá-/lox mice also showed decreased pulmonary arginase activity, indicative of a reduction of AAMs. RNA transcript analysis of isolated alveolar macrophages showed a strong association with promoting inflammation in LysMCreIL-4Rá-/lox mice. Together these data demonstrate that IL-4Rá-responsive macrophages control pulmonary inflammation and play an important protective role in the lung following N. brasiliensis infection.

Includes abstract. Includes bibliographical references (p. 109-125).

Sudden unexpected death in the young (SUDY) is the tragic fatality of seemingly healthy individuals aged between one and 40 years. Little is known about the demographics and risk factors of these cases at Salt River Mortuary (SRM), Cape Town. Therefore, this project aimed to retrospectively investigate the burden and profile of SUDY cases admitted to SRM, between 1 January 2016 and 31 December 2018. Of the total 11 588 cases admitted over this period, 833 (7.2 %) were SUDY cases, wherein males comprised the majority (64.3 %). Individuals were a median age of 31 ± 10.3 years at death, and the main location of death was ‘residential' (43.5 %). There were also significantly more males than females in the age category of 31 - 40 years who were found outdoors compared to all other locations (p < 0.001). Risk factors included physical activity, substance abuse, and co-morbidities with concomitant use of chronic medication. More than a third of individuals experienced breathlessness prior to death (45.0 %). Of cases with a confirmed natural cause of death, the main organ systems involved were pulmonary, cardiovascular, central nervous system and gastrointestinal, which parallels international trends. Akin to local studies, in analogous amounts, TB and pneumonia were the leading causes of death. Additionally, 21.1 % of cases were identified as candidates for genetic testing which may resolve undetermined cases or elucidate underlying predisposing factors to sudden death. Fortunately, 81.8 % had biological samples available for these retrospective analyses. Cases often had missing documentation which advocates for training to ensure compliance to standardised procedures. This study shows that males aged 31 ± 10.3 years with pulmonary and cardiac-related co-morbidities are the most vulnerable for SUDY whilst sleeping. Awareness interventions targeted at this population are thus needed in an attempt to reduce these tragic fatalities.

Background: Pregnancy at advanced maternal age (AMA) is associated with an increased risk of aneuploidy. In the Western Cape's public health sector maternal age alone is widely used to screen women for high risk of pregnancies affected by aneuploidy. The weekly pregnancy counselling clinic (PCC) at Groote Schuur Hospital (GSH) offers genetic counselling (GC) for women who are of AMA to inform them about their age-related aneuploidy risk, offer invasive diagnostic testing (IDT) and discuss the option of voluntary termination of an affected pregnancy. A recent audit at GSH showed that the uptake of IDT was low and other literature reports that South Africans tend to have a conservative view regarding termination of pregnancy (TOP). This study sought to understand what women expect from the GC service at PCC as well as what their experiences are of the service. Methods: This qualitative phenomenological study used a pragmatic approach and participants were recruited through purposive sampling. Semi-structured, in-person interviews were conducted after women had completed their GC sessions at PCC. Thematic analysis was used to analyse the data. Results: The results of this study suggest that participants (n=7) received very little information about their GC appointments at referring clinics, and that they generally did not have prior knowledge about age-related aneuploidy risks. Finding out about the age-related risk of aneuploidy was an emotional experience for the participants, but other factors, including normal ultrasound results, provided relief. The participants' choices regarding IDT and attitudes towards TOP reflected that of available literature as the uptake was low and most participants reported that they would not consider a TOP. The women reported that they would use the knowledge they gained during GC to educate other women in their communities about the pregnancy risks associated with increased maternal age. Generally, the participants believed that GC was useful and appreciated the opportunity. Conclusion: The participants in this study had limited health literacy and knowledge regarding AMA risks and GC. As a result, participants had no expectations of GC. However, the participants felt that GC was useful in helping them prepare for the possibility of a child with DS, and generally viewed the service in a positive light. Additionally, this study's results suggests that there is a need to educate women in local communities regarding AMA pregnancy risks.

We investigated immune responses beyond conventional T cells in the context of BCG vaccination and tuberculosis disease.

Studies have shown that the protection against L. mexicana infection is potentiated by a type 1 response, driven by IL-12 production by dendritic cells. In contrast, IL-4 and IL-13 cytokines are associated with susceptibility to L. mexicana causing cutaneous leishmaniasis. This has been demonstrated in studies involving BALB/c mice deficient in IL-4, IL-13, and IL-4Rα infected with L. mexicana. To determine the specific cell in IL-4Rα-/- BALB/c mice that contribute to the control of L. mexicana infections, studies on cell-specific IL-4Rα deficient mice need to be investigated. IL4Rα-CD4+T deficient mice revealed sex-dependent protection from L. mexicana infection, suggesting the critical role of non-lymphocyte cells in conferring protection against L. mexicana amastigote infection. Macrophage/neutrophil-specific IL-4Rα deficient mice are protected from L. major infection in the footpad. Surprisingly, this mouse strain infected in the base of the tail failed to control L. mexicana amastigote infection. Nonetheless, IL-4Rα-DC deficient mice were hyper-susceptible to L. major infection. This conundrum suggests that different Leishmania species, site of infection, and developmental stages of parasite dictate the outcome of the disease. Here, mice with a deficiency of IL-4Rα signaling on DCs and macrophage/ neutrophil cells were subcutaneously infected with L. mexicana promastigotes in the footpad, and skin lesion progression was measured, and the clinical phenotype was evaluated by investigating both humoral and cellular immune responses. Mouse strains had similar footpad lesion progression, parasite loads, humoral responses, expansion of CD4+ and CD8+ T cells, their activation, memory phenotypes, and infiltration of DCs, macrophages, and neutrophils into the lymph nodes compared to their littermate IL-4Rα-/lox controls. Interestingly, IL‐12p70 and IL‐10 produced by BMDCs and BMDMs were similar. Nevertheless, nitrite/urea production was not affected. Together, this study suggests that, unlike L. major, IL-4Rα signaling on DCs and macrophage/ neutrophil cells does not contribute to the susceptibility or resistance to BALB/c mice to infection with L. mexicana.

In 2020, the World Health Organization (WHO) reported 1.5 million tuberculosis (TB)- associated deaths and an incidence of 10 million new cases. The causative, Mycobacterium tuberculosis (Mtb), evades host immune responses by skewing macrophage polarization towards a less microbicidal alternative state to avoid classical effector killing functions. However, the molecular details underlying these evasion mechanisms remain incomplete and current therapy is challenged with drug resistance. Host-directed therapy (HDT) has recently gained attention, with long non-coding RNAs (lncRNAs) as potential targets due to their emerging roles in pathogenic immune responses. We previously performed cap analysis gene expression (CAGE) transcriptomics on IFN-γ stimulated (classically activated) and IL-4/IL-13 stimulated (alternatively activated) mouse macrophages, identifying 151 differentially expressed lncRNAs following Mtb infection. We validated the top 11 differentially expressed lncRNAs and two were chosen for this study, lncRNA-125, whose expression was regulated at different levels unstimulated and in response to IFN-γ and IL-4/IL-13, and lncRNA-612 whose expression was only induced by IFN-γ stimulation. Interestingly, the expression of lncRNA125 and lncRNA-612 was downregulated following Mtb infection. Therefore, this study aimed at functionally validating these lncRNAs in unstimulated, IFN-γ and IL-4/IL-13 stimulated and/or Mtb-infected mouse and human macrophages by a loss-of-function approach using chemically engineered antisense oligonucleotides (gapmeRs). Knockdown of lncRNA-125 by gapmeRs reduced Mtb growth and anti-inflammatory cytokine production mediated by increased apoptosis, nitrite and pro-inflammatory cytokine production in IL-4/IL-13 prestimulated mouse macrophages. Whereas knockdown of lncRNA-125 in IFN-γ pre-stimulated mouse macrophages favoured Mtb growth and anti-inflammatory cytokine production, with reduction of apoptosis, nitrite and pro-inflammatory cytokine production. Therefore, indicating that lncRNA-125 regulates macrophage polarization during Mtb infection. Knockdown of lncRNA-125 in human macrophages resulted in reduced Mtb growth and increased proinflammatory cytokine production in unstimulated, IFN-γ and IL-4/IL-13 pre-stimulated BMDMs infected with Mtb. Comparatively, gapmeR knockdown of lncRNA-612 reduced Mtb growth and increased pro-inflammatory cytokine production in IFN-γ pre-stimulated mouse and human macrophages. In mouse macrophages, these responses were mediated by increased apoptosis and nitrite production, with reduced anti-inflammatory cytokine production. Overall, these findings highlight lncRNAs as novel host factors to be further investigated as targets for TB diagnostics and adjunctive HDTs.