Ph.D. / Despite nearly three decades of intensive research, the HIV/AIDS pandemic remains a major challenge to modern medicine. The discovery and development of antiretroviral agents acting against various essential viral processes and enzymatic targets have greatly enhanced the quality of life for infected individuals, but no cure or preventative vaccine is available as yet and HIV infection is currently considered irreversible. Furthermore, the emergence of viral resistance to every class and type of antiretroviral treatment agent necessitates the continued discovery of antiretroviral agents with novel mechanisms of action. The first antiretroviral agent targeting the retroviral integrase enzyme (InsentressTM, Raltegravir) received regulatory approval from the United States Food and Drug Administration during 2007, validating HIV-1 integrase as a therapeutic target and providing a much-needed second- or third-line treatment option for treatment experienced patients. This enzyme was selected as a target for the current work. As limited data were available on the primary and secondary structure of the biologically relevant HIV-1 integrase enzyme, a first step in the present work was the construction of monomeric, dimeric and tetrameric models of the enzyme with biologically relevant catalytic centres incorporating both viral and host co-factors and DNA. The models were constructed to identify potential inhibitors of the strandtransfer reaction of HIV-1 integrase and were based on observations and interactions reported in the literature and on crystal structure data of HIV-1 integrase sub-domains and related structures available in the Protein Data Bank. The monomeric model was used as the macromolecular target in docking studies with “drug-like” compound databases, identifying the pyrrolidinone compound class as an in silico hit candidate for further development. Initial activity screening of a number of commercially available pyrrolidinone analogues against recombinant HIV-1 subtype B integrase in direct enzyme assays confirmed the predicted potential for strand transfer inhibition of the compound class, and provided initial support in the further development of this compound class as inhibitors of HIV-1 integrase that target the strand-transfer step. Retrosynthetic analysis of the pyrrolidinone hit candidates provided a facile one-pot, three-component synthetic pathway from readily available starting materials, which generally gave the proposed products cleanly and in acceptable yields. A range of closely related analogues were designed and synthesised. The analogues making up this series generally differed by only one functional group, in order to enable initial structureactivity relationship investigations during later stages of the project. Foreword Page XVI The synthesised pyrrolidinone analogues were screened through a range of direct and cell-based in vitro assays to determine the toxicity and strand-transfer activity of each. In general, the pyrrolidinone compounds proved well-tolerated in PM1 cell culture, with clear potential to further develop the strandtransfer inhibition of the compound family in second- and further-generation optimisation stages. Furthermore, the aqueous solubility and membrane permeability of each compound were determined in vitro, providing initial biological profiles of each test compound. As no in vivo testing was performed with any of the compounds during this first round of drug discovery and optimisation, several computational models were employed to extrapolate the in vitro and structural data to possible in vivo scenarios. Two pyrrolidinone analogues (11.6 and 15.2) were identified as low micro-molar strand-transfer inhibitors of wildtype-equivalent HIV-1 integrase, with low toxicity in cell culture and favourable solubility and permeability profiles. Resistance screening of these two compounds against four mutant HIV-1 integrases (Q148H; Q148H/G140S; N155H and N155H/E92Q) has shown some promise, with compound 15.2 retaining a measure of activity against the Raltegravir-resistant N155-mutants. These hit candidates will form the basis of structure-activity relationship optimisations in second- and further generation design stages.

R5-tropic HIV-1 is predominantly transmitted during unprotected sexual contacts, rendering CCR5 antagonist as an attractive agent not only for antiretroviral therapy but also for prevention. Here, we report two 1,3,3,4-tetrasubstituted pyrrolidine embodied compounds, TD-0232 and TD-0680, as novel small molecule CCR5 antagonists and investigate their specificities, potencies and underlying mechanisms. We found that both TD-0232 and TD-0680 inhibited a diverse group of R5-tropic HIV-1 and SIV strains in both single-cycle infectivity assays and live viral PBMC assays. When compared to other CCR5 antagonists, such as TAK-779 and the only FDA-approved Maraviroc, TD-0680 displayed the highest potency with EC50 values at the subnanomolar levels (range 0.09nM-2.29nM). TD-0232 and TD-0680, but not Tenofovir, a nucleoside reverse transcriptase inhibitor, completely blocked envelope-mediated cell-cell fusion and subsequent viral transmission. Critically, TD-0680 was potent at inhibiting the replication of a TAK-779/Maraviroc-resistant HIV-1 variant in PBMCs at a subnanomolar concentration. Interestingly, despite binding to a similar transmembrane pocket of CCR5, TD-0232 and TD-0680 functioned differently as revealed by site-directed mutagenesis and drug combination assays. Based on the sequence homology, we constructed a CCR5 molecule model using the crystallized CXCR4 as a template. By docking of CCR5 antagonists with CCR5, we identified a unique binding mode of TD-0680, which has not been described previously. TD-0680, with an exo-configuration, extended its interaction with the ECL-2 region of CCR5 in a protruding manner, thereby interrupting the interaction between the virus and its co-receptor more effectively. In an antibody recognition assay, we confirmed that TD-0680 had an enhanced inhibitory activity against the anti-ECL2 monoclonal antibodies binding. Furthermore, we investigated the antiviral activities of TD-0232 and TD-0680 that were formulated into a thermo-reversible acidic microbicide gel. Both drugs were stable in the acidic gels and could be released rapidly for long lasting and potent antiviral activities. Although human semen could enhance the infection of HIV-1, it did not seem to affect the potencies of the TD-0232 and TD-0680 gels. In summary, our findings suggest that TD-0232 and TD-0680 can be further developed not only as anti-HIV-1 agents for therapeutic purposes but also as potent microbicides for the prevention of sexual transmission of R5-tropic HIV-1. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

The present study sought to explore the functional changes that occur in the vasculature of HIV positive participants of African origin in Mthatha district of South africa which might lead to increased risk in their cardiovascular system. Available literature shows that arterial stiffness plays an important role in cardiovascular events such as stroke, vasculitis and myocardial infarction. Measurement of (aortic pulse wave velocity; PWV) provides some of the strongest evidence concerning the prognostic significance of large artery stiffening. This study was aimed at investigating the relationship between anthropometry, age, E-Selectin level, cytokine levels, haemodynamic variables, blood counts and blood lipid profile with pulse wave velocity. Some traditional cardiovascular risk factors such as alcohol, and smoking were also taken into account. This was a cross-sectional study comprising of 169 participants (62 males and 107 females). 63 were HIV negative (group A), 54 HIV positive on treatment (group B), and 52 were HIV positive not on treatment (group C). Pulse wave velocity (PWV) was assessed using the Sphygmocor Vx. Statistically, ANOVA was used for variables with normal distribution and non parametric tests were used for variables with skewed distribution. Notable significant differences were seen in the means of the following variables across all the 3 groups. Conclusion: This study showed that HIV infected patients with or without antiretroviral therapy have increase arterial stiffness which is associated with an increased cardiovascular risk. The sphygmocor is an accurate, non invassive and useful tool in the evaluation of arterial stiffness and its use in clinical practice should be encouraged. PWV and the augmentation index (AIx) are the two major non- iv invasive methods of assessing arterial stiffness. Life style modification should be incorporated into the management of HIV patients so as the continuous monitoring of their haematological and lipid profile.

M.Tech. (Homoeopathy) / Please refer to full text to view abstract

D.Phil. / This enquiry represents an attempt to understand the ways in which the ecology of ideas surrounding HIV and Aids in post-apartheid South Africa functions discursively to silence people living with the dis-ease. In this regard, it seeks to understand how the range of subject positions available to people with HIV and Aids influences their opportunities for treatment and disclosure. The meanings emerging from this enquiry have implications for interventions aimed at people living with HIV and Aids, in that they challenge the liberal humanism underpinning a Western individualist paradigm which constructs people as ‘rational’ and ‘responsible’ on the basis that such constructions tend to attribute guilt or moral culpability to people living with HIV and Aids. The conversations and narratives elicited in the process of this enquiry suggest that such discourses constitute a form of disciplinary power in a Foucauldian sense, positioning people living with HIV and Aids defensively and limiting their options for ‘positive’ self-definition by foreclosing available subject positions, thereby contributing to the spread of HIV and Aids. Hence, this enquiry focuses on social constructions of morality and the impact of these on participants’ attempts to maintain key relationships that afford them a ‘positive’ sense of them-selves. Thus, it looks at experiences of connection and dis-connection and explores the ways in which efforts to retain ‘relatedness’ in order to maximise possibilities for the co-construction of a ‘moral self’ mediate opportunities for disclosure and treatment options. The enquiry aimed to assist participants in deconstructing dominant social constructions of HIV and Aids implicit in cultural and political discourse by applying a critical, poststructuralist and discourse-analytic lens in order that they might resist moral attributions based on liberal humanism and access their own voices in narrating the experience of living with HIV and Aids in keeping with their lived experience. My aim in this regard was to resurrect alternative or previously silenced accounts and to open up spaces for a multiplicity of meanings associated with HIV and Aids to emerge and be heard, toward the end of breaking the silence and creating a conversational space in which people’s meanings could simultaneously be heard and challenged through dialogue.Ultimately, this enquiry highlights the importance of attempting to understand the local and idiosyncratic nature of people’s constructions of HIV and Aids, which are often a hybrid mix of ideas and meanings circulating within social, cultural and political discourse. It also underscores the salience of considering people’s lives in context and particularly their need to maintain relationships that afford a positive sense of self. This is reflected in the tendency for participants to construct their identities in relation to significant others and for these relationships to mediate decision making in relation to HIV and Aids by availing or foreclosing certain subject positions, depending on the discourses within which they are situated.

M.Tech. / HIV/AIDS is one of the greatest and still unresolved challenges facing the world today (Giese, 2002). The UNAIDS (2004) Reports, 38 million people are infected globally with HIV/AIDS. One of the most devastating impacts of HIV/AIDS is the effect on children. Over 5 million infants have been infected with HIV/AIDS since the beginning of the epidemic and 90% of these cases are in Africa (Osborne, 2002). The aim of the study was to determine the effect of a combination of four homoeopathically prepared growth factors, compounded and named Cell Signal Enhancers®, on CD4% and CD4+ cell count, growth parameters such as weight, height and head circumference and clinical outcomes in children with HIV/AIDS. A sample of thirty-one (n=31) HIV positive children between one to thirteen years of age was recruited. Twenty-five (n=25) participants completed the trial. The participants were recruited from an informal squatter area called Finetown, situated south of Johannesburg. The parents or legal guardian of each participant were required to read and sign a Patient Information and Consent form (Appendix A). The duration of the study was fourteen weeks. Each participant acted as his/her own control in a two week pre-treament period. Analysis of CD4% and CD4+ cell count, measurements of growth parameters that included weight, height and head circumference and evaluation of the clinical outcomes were conducted pre-treatment, during treatment and post treatment. The school principal and daily caregiver administered the homoeopathic growth factor medication to each participant. One tablet was given three times a day, for a twelve week period. Statistical models such as a paired t-test, one-sample t-test and linear regression were used to analyse the data. The resultant analyses of the data have provided the following conclusions. HoGF treatment improved immune function of the participants, as there were increases in CD4% and CD4+ cell counts and an overall decrease in frequency of HIV symptoms. HoGF intervention reversed growth failure. This was demonstrated with increases in weight, height and head circumference that resulted in a form of “catch up” growth. When compared to published data trends of age-matched subjects using ART, HoGF demonstrated more favourable effects in the CD4%, CD4+ cell counts and growth parameters in a twelve week period. HoGF treatment was effective in each stage of HIV/AIDS; namely the asymptomatic, symptomatic and AIDS group. HoGF has proven to be effective in treating HIV infected children living with limited resources. It showed a 52% positive result as statistically significant versus a 5% prediction by random chance. Statistical significance was detected in the following; height, head circumference, energy, strength, vomiting, lymphadenopathy, skin lesions, respiratory tract infection, sinus tenderness and throat infection. There were no reported signs of adverse side effects while on HoGF treatment. The results of this study are expected to initiate further, much needed research in the area of HIV/AIDS and homoeopathy in both children and adults. It is recommended that future studies include a control group with placebo for inter-group comparisons. HoGF treatment can be seen as a possible public health option for treating HIV/AIDS in South Africa.

Indiana University-Purdue University Indianapolis (IUPUI) / HIV-1 infection is one of the greatest public health concerns today. The current HIV/AIDS therapy is effective in halting virus multiplication and has improved the outlook of AIDS; however, high cost, side effects, and the rise of drug-resistant viral strains have posed challenges for long-term treatment and management and mandate development of alternative anti-HIV therapies. Despite the fact that a great deal of progress has been made in our understanding of the infection over the last twenty-seven years, there are many unanswered basic scientific questions and no vaccines. In this study, we focused on two aspects related to the HIV-1 protein Nef: one is development of a Nef-based anti-HIV therapeutic strategy; the other is discovery of a novel mechanism that accounts for Nef-enhanced viral infectivity. We first devised an anti-HIV therapeutic strategy that took advantage of the high virion incorporation of the Nef mutant Nef7 to deliver anti-HIV factors to the virion. We performed a series of proof-of-concept experiments, using the host anti-HIV cellular factor APOBEC3G (A3G). The Nef7.A3G fusion protein retains important properties of Nef7: higher virion incorporation efficiency, lack of PAK2 activation, and reduced CD4 and MHC I downregulation, as well the anti-HIV infectivity function of A3G. Moreover, virus-like particle (VLP)-mediated delivery of Nef7.A3G into infected CD4+ T lymphocytes leads to inhibition of HIV-1 replication in these cells. These results support the use of Nef7 as an anti-HIV therapeutic strategy for the delivery of therapeutic proteins into HIV-1 virions. HIV-1 Nef protein has long been known to enhance viral infectivity. However, the underlying molecular mechanism remained elusive. Here we show that Nef is important for VS formation and VS-mediated virus transmission from cell to cell, especially in primary cells. Nef accomplishes this by inducing the clustering of VS components CD81 and ZAP70 and by inducing formation of actin protrusions, and these functions involve specific and distinct Nef domains. These findings not only yield new insights into the regulatory function of Nef in viral infectivity, but could also lead to development of more effective anti-HIV therapies that work equally well at blocking both VS-mediated and cell-free virus infection.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: While antiretroviral therapy decreases HIV/AIDS morbidity and mortality, long-term treatment results in insulin resistance and cardiovascular diseases. A possible cause of such adverse effects may be an increase in oxidative stress resulting from protease inhibitor (PI)-induced mitochondrial dysfunction. We therefore hypothesized that PI treatment, specifically Lopinavir/Ritonavir, results in increases in myocardial reactive oxygen species (ROS), leading to downstream outcomes, i.e. elevated apoptosis. Moreover, we proposed that increased ROS levels in this instance might occur as a result of PI-mediated induction of the non-oxidative glucose pathways (NOGPs). In light of this, we also investigated the effect of PI treatment on the NOGPs by employing both in vitro and in vivo samples. For the in vitro work we employed a rat cardiomyoblast cell line, while tissues (heart, liver) were collected from two separate experimental models, i.e. a) Group A exposed to PIs via mini-osmotic pump for a period of eight weeks, and b) Group B administered PIs via a jelly-based method for 16 weeks. We found that PIs increased mitochondrial ROS levels in vitro but that this was not accompanied by a parallel rise in programmed cell death. Moreover, we found no induction of the NOGPs in response to PI exposure (for both in vitro and in vivo models here employed). However, we found that the AGE pathway was significantly down-regulated in the liver of Group A. Investigation into a proposed mechanism for this observation proved inconclusive and further studies are thus required to clarify the significance in terms of metabolic dysfunction found in the Group A model. Our study thus shows that PIs can increase ROS levels (in vitro) but that compensatory antioxidant mechanisms may prevent this in vivo. Subsequently, downstream effects were limited i.e. we did not observe NOGP induction and programmed cell death. An intriguing finding emerged, however, i.e. that PIs can elicit an impact on the AGE pathway. We propose future studies with modifications to the current rat and cell models in order to evaluate the downstream effects of PIs on the NOGPs and programmed cell death. / AFRIKAANSE OPSOMMING: Terwyl antiretrovirale terapie MIV/VIGS morbiditeit en mortaliteit verlaag, veroorsaak langtermyn behandeling insulienweerstandigheid en kardiovaskulêre siekte. 'n Moonltike oorsaak van sulke newe-effekte kan 'n toename in oksidatiewe stres veroorsaak deur die protease inhibeerder (PI)-geïnduseerde mitochondriale wanfunskionering. Ons hipotetiseer dat PI behandeling, spesifiek Lopinavir/Ritonavir, versoorsaak 'n toename in miokardiale reaktiewe suurstofspesies (ROS), wat aanleiding gee tot afstroom uitkomste, i.e. verhoogde apoptose. Verder, stel ons voor dat verhoogde ROS vlakke in hierdie geval onstaan as gevolg van PI-gemedieerde induksie van die nie-oksidatiewe glukose weë (NOGWe). In die lig hiervan het ons ook die effek van PI behandeling op die NOGWe ondersoek deur beide in vitro en in vivo monsters te gebruik. Vir die in vitro werk het ons van 'n rot kardio-mioblastsellyn gebruik gemaak, terwyl weefsels (hart, lewer) versamel is van twee afsonderlike eksperimentele modelle, i.e. a) Groep A blootgestel aan PIs via mini-osmotiese pomp vir 'n periode van agt weke, en b) Groep B PIs is toegedien via 'n jellie gebaseerde metode vir 16 weke. Ons het bevind dat die die PIs mitochondriale ROS vlakke in vitro verhoog maar dat dit nie vergesel is met 'n paralelle toename in apoptose. Verder is geen induksie van die NOGWe in reaksie op PI blootstelling waargeneem (vir beide in vitro en in vivo modelle). Hoewel ons het bevind dat die AGE weg in die lewer van Groep A beduidend afgereguleer is. Ondersoek na 'n moontlike megansime vir hierdie waarneming was onoortuigend en verdere ondersoek is nodig om die betekenis in terme van die metaboliese wanfunskionering in die Groep A model vas te stel. Ons studie toon dus aan dat PIs, ROS vlakke (in vitro) verhoog, maar dat kompensatoriese anti-oksidant meganismes in die hierdie in vivo model verhoed word. Gevolglik is die afstroom effekte beperk i.e. ons het geen NOGWe induksie en aptoptose waargeneem nie. 'n Interesante bevinding het wel uitgestaan, i.e. PIs kan 'n impak hê op die AGE weg. Ons stel dus voor dat toekomstige studies met modifikasies, tot die huidige rot- en sel-modelle gemaak word om die afstroomeffekte van PIs en apoptose te evalueer.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science Johannesburg, August 2012 / It is known that infectious agents elicit different responses in different individuals which strengthens the view that susceptibility and resistance to infectious diseases has a genetic component. These differences in susceptibility to disease can be observed in populations. APOBEC3G is a member of the cytidine deaminase gene family located on chromosome 22. It is crucial in non-permissive cells as it functions as part of the innate immunity system and is an inhibitor of the HIV-1 accessory protein vif. The goal of the study was to develop genotyping assays and estimate allele frequencies. Thus, genetic variation within APOBEC3G was identified and characterized in black South Africans. Indirect genotyping assays were designed to amplify regions within the upstream non-coding region, and in exon 4 of the coding region of the gene. Selected polymorphisms were then genotyped using allele-specific PCR, RFLP-PCR and Pyrosequencing™ assays. Reanalysis of sequence data from 2003 showed numerous SNPs were well represented. Comparison of sequence data at various SNPs showed that allele frequencies were similar to frequencies in other African populations. The only sequenced SNP that deviated from the frequencies in Ensembl was -590. Thus the sequencing was a useful tool for detection of variation. ASA proved to be the least reliable genotyping technique as the minor allele frequency of -571 (0.59) deviated from the published frequency of 0.894 in Africans. RFLP analysis proved more reliable for genotyping -571 and H186R. The minor allele frequency was estimated to be 0.84 and 0.32 for -571 and H186R respectively. The frequency of H186R is similar to published data from An et al (2004) and Reddy et al (2010). If SNPs are in LD they occur together on the same haplotype more often than by chance. Usually SNPs that are in LD are in close proximity. However our data suggests -571 and H186R SNPs which are 5kb apart are not in LD. A LD map of chromosome 22 shows highly variable pattern of LD (Dawson et al, 2002). Widespread regions of nearly complete LD up to 804 kb in length are intermingled with regions of little or uundetectable LD. Haplotype analysis showed the most frequent haplotype was GA. This was the most frequent haplotype when the sample types were subdivided according to spoken language. in comparison to studies from An et al, (2004) D’ of the two SNPs was estimated at 0.967. The linkage disequilibrium (LD) revealed a non-independence of allele segregation because the loci analyzed were strongly linked in the Apobec 3 G gene. The data are consistent with greater genetic diversity of African populations and can form the basis for further evaluation of the role of variation in this gene in response to HIV.

The 2013 case report of the “Mississippi baby,” who was started on antiretroviral therapy (ART) within 30 hours of life and maintained off-treatment remission for 27 months before HIV was once again detectable, generated renewed interest in the benefits of early ART, as well as optimism that HIV remission is a possibility if ART is started very early. The overarching goal of this dissertation was to advance our understanding of the relationship between early ART and three outcomes – the possibility of HIV remission, improved viral outcomes, and epigenetic changes – in HIV-infected infants and young children. First, a systematic review was conducted to assess current published data in support of the possibility of very early ART leading to HIV remission in infants. Evidence from this review suggested that although early ART does appear to be associated with better sustained virological control and smaller reservoir size, there are limited data at this time to support a strong link between early ART and HIV remission. Second, the association between age at ART initiation and virologic outcomes after ART initiation was empirically assessed using data from five cohorts of HIV-infected infants and young children initiating ART before 2 years of age in Johannesburg, South Africa. In three cohorts, there was no consistent evidence of an association between early ART initiation and rates of initial viral suppression. However, there was a consistent benefit of early treatment initiation on long-term viral control in two cohorts. Finally, an epigenome-wide association study was conducted to identify differential DNA methylation patterns between ART-treated HIV-infected and HIV-uninfected South African children. A total of 1,309 differentially methylated CpG sites associated with HIV status were selected (FDR q-value <0.05; |Δβ| >0.05), after adjustment for age, sex, and cell type proportions. In addition, 315 differentially methylated regions associated with HIV status were selected (Stouffer p-value <0.05, maximum |Δβ| change in the region >0.05, and containing at least two or more CpG sites). Many of the genes identified in both the site and region approaches were located in the major histocompatibility complex (MHC) region on chromosome 6, a region that plays an important role in the adaptive immune system. This novel study provided evidence of an association between perinatally-acquired HIV infection and a large number of changes in DNA methylation in school-aged children on ART, and highlighted potential new lines of investigation into the biologic pathways influenced by HIV and its treatment. Overall, this dissertation increased our understanding of the timing of early ART initiation in HIV-infected infants and addressed gaps in our knowledge relevant to outcomes associated with early ART initiation. As public health practice continues to move towards infant diagnosis of HIV at birth and early life initiation of ART, these findings help to inform future research that will guide HIV care in infants.